BioAP 3160 - LECTURES 1-5 January 23-February 1, 2012

Different cell organelles : scheme of a typical cell - How to recognize their ultrastructural

appearance - composition, biosynthesis, and function(s) of organelles and of their

components: working our way out from the nucleus.

THE NUCLEUS

In most cells the most prominent organelle - Repository of genetic information -most cells

have nucleus [exceptions : erythrocytes (they do in birds !), platelets]

- Distinguishing feature between procaryotes and eucaryotes

- The nuclear envelope: separates nucleoplasm & cytoplasm

-nuclear envelope is a double membrane

-has an inner membrane and outer membrane

-inner membrane functions are related to nuclear fxn

-outer membrane is continuous with the cisternae of the ER

- Nuclear pores allow exchange of materials (information) between nucleus and cytoplasm -

Why is separation needed between nuclear material and cytoplasm?

-need separation so that transcription and translation are segregated

What are the main physiological implications?

-allow the many proteins involved in replication and transc. to be concentrated where they are

needed, keeps nuclear enzymes separate from cytosolic enzymes,

- the nucleolus, (Appears dark in EM because of concentration of ribosomes and rRNA being

made), the regions of condensed (heterochromatin) and dispersed (euchromatin) chromatin.

- variation in the amount of nuclear material/cell- Multinuclearity, polyploidity, politeny, gene

amplification

- Variability in shape of the nuclei - often has physiologic significance

- Differences in the relative amounts of heterochromatin/euchromatin;

Constitutive vs facultative heterochromatin – const. hetero is always there, like centromeres,

telomeres, etc. Facultative is like maternal/paternal imprinting

- C-value paradox : amount of DNA/haploid nucleus does not correlate with organism

complexity - It is now assumed that in mammals only about 1% of the DNA is involved directly

in RNA synthesis (classic genes)

Chromatin = DNA + associated proteins = most of content of nucleus

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- minimal linear chromosome elements: DNA replication origin (const. hetero. Sequences that

don’t correspond to active genes but can bind to components of replication apparatus),

centromere (const. hetero. Where mitotic spindle attaches), 2 telomeres (repetitive sequences

necessary to prevent shortening of chromosome after replication)

- 1 molecule double stranded DNA per chromosome (ALWAYS EXIST, even during interphase)

- in humans max length: 10 cm (2-3x108 bp)

- DNA-binding proteins in eukaryotes: histones & non-histone chromosomal proteins

Histones: about 60% of protein in the nucleus, total mass of histones about equal to that of DNA

- highly conserved, relatively small, high proportion of positively-charged AAs (Lys, Arg)

- 5 types of histones: nucleosomal histones (H2A, H2B, H3, H4), H1 histones

- H3 & H4 among the most highly conserved proteins

- H1 less conserved, various types in each nucleus - Cooperative binding

- Histones package DNA - also contribute to gene regulation

- -All histones’ core regions very similar, differences at C and N termini

- Modifications : H1 phosphorylation (serine) –contributes to condensation

H3,H4 acetylation (lysine) - less condensed chromatin (DNAse I sensitivity)

Methylation –modification controls condensation and expression

- methylation of H3 causes hetero. Development and gene silencing

Ubiquitin-H2A, only in interphase, active genes

- All of the above possible because the octameric proteins all have tails that stick out of

octamer that get modified

Non Histone Proteins:

-compose about 1/3 less than histones of total protein in cell

- defined as proteins leftover when histones have been removed from cell

- very heterogeneous

- less basic than histones

- Three possible functions: 1) structural components of chromatin involved with higher order

compaction of nucleosomes, 2) enzymes involved with DNA replication, RNA transcription,

histone modification, RNA processing 3) control of gene expression with specific recognition sites

on DNA – zinc finger

Sequence-specific DNA-binding proteins: DNA folding, initiation of DNA replication, control of

gene transcription; Bind to DNA without disturbing the double helix

- most gene-regulatory proteins bind to the DNA major groove

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- helix-turn-helix motif common feature of DNA-binding proteins (example, homeodomain

proteins)

- zinc fingers (zinc forms complex with Cys and His and creates fingers that fit into major

grooves), leucine zipper, helix-loop-helix

- Cooperative binding

- Other non-Histone chromosomal proteins: Enzymes, packaging proteins, transcription

factors

STRUCTURE OF CHROMATIN : in interphase little can be seen by conventional TEM

Nucleosome model : -compaction and condensation of DNA to fit into nucleus = nucleosome

- one nucleosome is linker DNA, histone core, and 146 bp around core

- basic informations leading to it: Miller spreads (floating chromatin on air/water interphase,

beads on a string appearance (11 nm diameter); if you use pure DNA, just get string and no

beads ->> beads must be proteins = histones

Miller spreads on saline : 30 nm nucleoprotein fiber – this demonstrated the first level of

organization, but still need two more to fit DNA in nucleus ->> 3 stages of DNA packaging

- extensive packaging required to condense 5 cm DNA molecules into nuclei 5 µm in diameter

- Micrococcal DNAse digestion : at low concentration DNA fragments of about 200 bp - at higher

concentration, 146 bp fragments:

- smallest pieces of digested DNA on gel electrophoresis were 205 bp, tells us that there are

pieces of DNA 205 bp long that cannot be digested – DNA wrapped around histone core is

protected from digestion.

- if you add higher conc. Of DNAase, take out the histone core and the linker DNA, get

fragment 146 bp long. So, linker region = about 56bp

- nucleosome beads produced by digestion with micrococcal nuclease

Nucleosome model: octamer of H2A,H2B,H3,H4 - 146 bp DNA wrapped on it

- Histone H1 NOT part of octamer, it binds to the outside of the core, kind of seals the coil of

DNA around the octamer core.

- linker DNA (0-80 nucleotide pairs)

- nucleosomes not randomly positioned: propensity of DNA helix to form tight loops (sequence-

dependent);

DNA-binding proteins (DNase I, nuclease-hypersensitive sites)

- Histone H1 (six closely related varieties) packages nucleosomes into the 30 nm fiber; higher

order structures; cooperative binding. Regulated by phosphorylation. When phosphorylated

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chromatin becomes more compacted.

- 11 nm fiber is linker, bead, linker, bead

- 30 nm fiber is beads on a string wrapped around itself: still don’t know its structure for sure

- RNA synthesis in the presence of histones

CHROMOSOMES : visible during mitosis - number & shape characteristic for each species (23 in

humans) –present all the time (duh)

- Unitemy : 1 molecule of double stranded DNA/chromosome

- At least two more orders of compaction than in nucleoprotein (30 nm) fibers

- Highly condensed mitotic chromosomes

- examples visible under the microscope: lampbrush chromosomes (amphibian oocytes,

meiotically-paired chromosomes covered with RNA-protein complexes) & polytene

chromosomes (Drosophila salivary cells, 1024 strands of identical chromatin, light & dark

bands; chromosome puffs)

- transcriptionally-active chromatin is less condensed

- nuclear matrix may help organize chromosomes/chromatin in interphase nuclei

- Condensation of chromatin in mitotic chromosomes (phosphorylation of histone H1, sister

chromatids held together at centromeres; transcriptionally inactive)

- chromosome bands (G bands=A-T rich; R bands=G-C rich)

- in order to decondense nucleosomes and access DNA for replication, transc. and gene

expression, use remodeling complex A: starts unwinding process and allows DNA binding (non

histone) proteins to bind and hold DNA open, then remodeling complex B dissociates the DNA

binding proteins and the nucleosomes are allowed to reform.

- during this process scaffold proteins remain bound to DNA (keep it straightened out)

HV-TEM view of chromosomes : Looped domains in chromosome spreads; In interphase,

chromatin of each chromosome dispersed in the nucleus, and intermingled, and MUCH LESS

CONDENSED

Bands in chromosomes - regions of different composition, covering much more than a single

gene

- bands are NOT individual genes!

- Bands tell us that some regions of a chr. are more or less tightly wound

- CHROMOSOMES NOT UNIFORM DOWN THEIR LENGTH

- Banding helps us match chromosomes during karyotyping

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DNA REPLICATION

- higher eukaryotes always have >1 replication origin

- takes place during the S phase of the cell cycle

- The replication apparatus : studied mostly in bacteria (mutants) but assumed to be

essentially the same in eukaryotes (however, histones are present in this case !). Accurate,

rapid synthesis of DNA (500 nucleotides/sec in bacteria, 50/sec in mammals)

- DNA strands function as templates - replicating fork; continuous synthesis in the 5'-3'

direction (more accurate synthesis - proofreading - tautomeric forms of bases)

Accepted model of DNA replication

- DNA replication fork is asymmetrical (leading strand, lagging strand, Okazaki fragments)

- 1 DNA polymerase in bacteria, at least 2 in eucaryotes:

- Polymerase alpha has primase subunit that synthesizes primers. Alpha is too slow to

synthesize by itself so switch to polymerase delta, much faster enzyme. For leading strand this

switch only happens once. Happens more on lagging (switching from primers much more often)

- In order to switch from alpha to delta need PCNA (proliferative cell nuclear antigen). It’s also a

target for protein kinase-inhibitors – slows/stops DNA rep to allow repair to occur

- RPC (replisome progression complex) binds to DNA on lagging strand, and PCNA binds to

RFC, and PCNA helps remove pol-alpha and replace with pol-∂ (switching);

- accessory proteins: RPA replication protein A = SSB protein that stabilizes the template strand

- RFC replication factor C= stabilizes the lagging strand while PCNA binds and switches alpha

out for delta

- Proofreading mechanisms: tautomeric forms of the four DNA bases, changes in helix geometry

3'-to-5' proofreading: exonuclease activity of DNA polymerases

- ATP driven helicases: unwind the template DNA helix at replication forks, use ATP to

perform fxn, most of the time are bound to inhibitor but get called over by initiator protein binding

to replication origin and lose inhibitor during repl.

- Single-strand DNA-binding (SSB) proteins: prevent formation of hairpin loops

- "Clamp" proteins keep DNA polymerases attached to DNA while they are moving

- DNA Primase synthesizes short (about 10 nucleotides) RNA primers on lagging strand - DNA

polymerase synthesizes 200 (eukaryotes) or 2000 (bacteria) nucleotides-long stretches of DNA

(Okasaki fragments) - RNA primers digested away, empty spaces filled in, DNA ligase seals

nicks

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- Winding problem (one complete turn about the axis of the parental double helix every 10 base

pairs)

- solved by topoisomerases: topoI opens up one strand of DNA by utilizing a tyrosine active site

and covalently attaches to DNA phosphate, breaking phosphodiester linkage. (Energy is retained

in bond with tyrosine). Once bond is broken, DNA can rotate around other unbroken and relieve

tension. The strand of DNA is then re-formed with retained energy.

Replication forks (special sequences of DNA present at their center) – Replication forks can be

unidirectional or bidirectional (usually bidirectional)

- Replication bubbles/replication origins (up to 300 nucleotides long), binding of initiator proteins,

then helicase, then primase – primosome = complex formed when initiator, helicase, and primase

are all bound to template DNA , then DNA polymerase can bind and begin synthesis based on

primer!

- well characterized in bacteria and in yeasts (ARS sequences)

- in higher eucaryotes replication origins activated in clusters (units)

- Different regions of each chromosome activated in a reproducible order during S phase

- Synthesis of new histones during S-phase of the cell cycle; packaging of new histones

- Special problem of the Telomeres (G-rich repeats added to chromosome ends by telomerase)

– Telomerase = DNA polymerase with bound RNA template: enzyme that adds nonsense bases

to 3’ end of parental strand (using its own RNA subunit as a template) so that DNApol can use

that as a template and make sure that replication of parental strand continues through all

necessary bases (from 5’ to 3’ end of new strand)

- DNA re-replication block (licensing factors)

TRANSCRIPTION : SYNTHESIS OF mRNA - highly selective process

Structure of a "typical" gene : Promoter - binding of eucaryotic RNA polymerases to protein-

DNA complexes (transcription factors) - enhancer elements, upstream sequences, TATA box

(TATA factor), silencers, position effects

- TATA box = seq. within promotor that euk. RNAP recognizes and transc. starts about 20 bp

downstream

- activator protein can bind to enhancer upstream of promotor to upreg. Transc. (repressor does

opposite)

- Genes in pieces : Introns and Exons.

- only about 1% of chromosomal DNA transcribed

- start/stop signals for transcription; stop signals (in E. coli): self-complementary region + run of

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Different types of RNA : at least three type of RNAP in euks!

Ribosomal RNA (rRNA) – made in nucleolus (except 5S), found in ribosome’s (80% all RNA)

Transfer RNA (tRNA) –isoaccepting, at least one per amino acid

Messenger RNA (mRNA)

-we think of mRNA as being most common, but is really only 3-5% of total RNA in cell

- 5’ caps (important for binding to ribosomes – translation), poly A tail (important for stability but

not present in all, absent in histones – why histones are only synthesized in S phase right before

they’re required)

- introns, exons, made into proteins by ribosomes

RNA polymerases - bind to promoter regions (specific DNA sequences, oriented, determine

which strand of DNA is used as template RNAP can work in either direction, but in reality only

works on one strand bc of polarity of promoter)

- bacterial & eucaryotic RNA polymerase complex (multiple subunits, regulation); some bacterial

viruses encode much simpler RNA polymerases

Biosynthesis of RNAs: opening of DNA strands, 5' to 3' direction of RNA synthesis (reads

template DNA 3’-5’)

One RNA polymerase in bacteria - different subunits, sigma factor, elongation factors

Three RNA Polymerases in eukaryotes: all require acc. Proteins to fxn

RNA Polymerase I - makes large ribosomal RNAs - unaffected by alpha-amanitin

RNA Polymerase II - makes mRNAs - very sensitive to amanitin

RNA Polymerase III - makes tRNAs, 5S RNA of ribosomes, and other small, stable RNAs –

moderately affected by amanitin

- additional initiation proteins required for binding of eucaryotic RNA polymerases to promoters

(Transcription factors) already bound to DNA

- Association of DNA with histones must be altered (they remain in nucleosomes during

transcription) DNA must be opened up somewhat, cannot use 30 nm fiber

- Different RNA Polymerases recognize different start signals

- RNA Polymerase III - requires 5S transcription factor - which recognizes DNA sequences in

the middle of the gene

- RNA Polymerase II transcribes different sequences at very different rates

- RNA synthesis visualized by TEM of spreads - globular RNA polymerase “Christmas tree”

- origin of replication on DNA is located right before where RNA strand is shortest (on strand

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being transc. by multiple ribo)

-termination signal is located just after longest RNA strand

- dots at end of RNA molecules = spliceosomes which shows that processing begins on 5’ end

before 3’end is synthesized

Stages of RNA synthesis : need ATP to start AND end transc.

1) interaction of RNA polymerase with DNA to form a binary complex;

2) Initiation of RNA chains;

3) Elongation (can be divided in at least 5 substeps);

4) Selective termination - requires accessory proteins, ATP.

- mRNA synthesis: heterogeneous nuclear RNA (hnRNA) is immediately bound to proteins

(hnRNP particles)

- all RNA except tRNA does NOT like to be naked, so as soon as it’s synth. It binds proteins

- formation of spliceosomes

- Capped at 5' end: 7'Me guanosine-PPP-A/G (will mediate binding to ribosomes, stabilizes

mRNA)

- Tailed at 3'end: poly-A tail added by poly-A polymerase (AAUAAA signal 20-30 nucleotides

upstream of cleavage: instructions to ADD tail are encoded, but actually poly A seq. not

encoded); transcription continues until termination signal reached; RNA fragment rapidly

degraded: signal to add Poly A tail comes before termination signal, so RNAP keeps transc. after

poly A tail has been added by PolyA polymerase, but any RNA after tail gets degraded

- poly-A tail aids in exit from nucleous, gives stability in cytoplasm, required for efficient

translation, poly A binding proteins bind to tail to confer stability

-polyA tail never added to histone mRNA’s because don’t need to leave nucleus, and need to be

unstable so they can be destroyed following S phase

- primary transcript (average length for mRNAs : 8000 nucleotides). Very unstable - reduced to

average length of 1200 nucleotides within 30 min

- mRNA processing (splicing, removal of introns)

- spliceosomes contains small RNAs that hybridize specifically to sequences to catalyze RNA

splicing (recognized donor-acceptor junctions), small RNAs involved (snRNPs), RNA/RNA

pairing, lariats = where RNA being removed loops out

- Alternative splicing of the same primary transcript can generate different proteins

- Changes in development, different tissues: depending on time in dev. Or which type of tissue

the cell is, different introns get spliced and different exons get put together, and you get different

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gene products

- Transport of mRNAs to cytoplasm delayed until splicing is complete and highly regulated by

nuclear pore acceptor complexes. Proteins associated with RNA change after entering

cytoplasm.

Posttranscriptional control of gene expression

1. attenuation (RNA synthesis) Transcriptional control

2. mRNA processing in the nucleus

3. retention in the nucleus (export to cytosol)

4. mRNA localization in the cytosol (signals in the 3' untranslated region) is polyA tail there?

5. translational controls

6. mRNA degradation in the cytosol – some have halflife of minutes, others stable for days

- can control gene expression after mRNA made by keeping mRNA in nucleus longer or forever,

not making it, keeping it isolated in cytosol, not translating it, degrading it, editing it, controlling

protein product

The NUCLEOLUS : by TEM recognizable as an aggregate of closely packed filaments (ribosomal

RNA), dense granules (almost finished ribosomes), amorphous matrix, associated chromatin

Site of Ribosomal RNA synthesis (not 5S RNA) by Pol I and assembly of Ribosomes

Multiple ribosomal RNA genes, arranged in tandem - "Christmas tree" appearance in spread

chromatin preparations: nucleolar constrictions are regions of DNA where there are hundreds of

copies of nuc. Org. regions. (need many copies to make many rRNAs quickly) and Christmas

trees result from rRNA being synth. At each copy simultaneously

- Nucleoli made up of nucleolar-organizer regions of chromosomes (1 or more)

- Larger RNA gene initial transcript that is processed to get ¾ rRNA genes. The 5S RNA gets

together with the other 3 along with ribosomal proteins that are made in the cytoplasm but

imported into the nucleolus

- Present on five chromosomes in humans (10 in diploid state)

- Nucleolar- Organizer regions of different chromosomes meet in same region of the nucleus

- the parts of the chr. that are nucleolar organizing coding regions (AKA DNA coding for rRNA)

are all sticking into same region of nucleus become the nucleolus

- no nucleolus in mitotic cells because the DNA is super condensed and no transc. of rRNA

taking place

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- Synthesis of tRNA's : Clove-leaf structure - about 100/cell in eukaryotes - Processing of

tRNAs packaging & processing of 45S rRNA precursor

- nucleolin: an abundant RNA-binding protein apparently only associated with rRNAs

- Overall scheme of ribosomes synthesis

- 5S rRNA is coded for and transc. outside of nucleolus (on chromosome 1), so must be

moved into nucleolus to be added to immature subunits as it’s assembled

- Small and large immature subunits leave nucleolus and become mature subunits in nucleus

- mature small and large subunits leave nucleus separately and assemble when NEEDED in

cytosol (when mRNA binds small subunit)

NUCLEAR ENVELOPE : 2 membranes about 20-40 nm apart, composition and function differ:

- Inner membrane organizes chromosomes, binds to fibrous lamina proteins;

- Outer membrane, similar and contiguous to ER, ribosomes are often present

- Perinuclear space in between, contiguous with lumen of RER

NUCLEAR PORES (nuclear pore complexes) : sites of exchanges between nucleus and

cytoplasm, about 9 nm aqueous channels

-pores are highly selective but small molecules (ions, ATP, Pi, etc) pass freely through

- test this using non-nuc. proteins (bc know nuc. has no mech. to transport them already)

-for prot. 1-2 nm, go freely; 2-3 nm, takes longer; 3-5 nm, never

Scheme of nuclear pore :

- 2 rings of 8 subunits each, one on cytoplasmic side and other on nuclear.

- 2 rings connected by 8 column subunits (bulk of the pore wall)

- In the center have 8 annular components (extend spokes toward center) where receptors are

- At cytoplasmic side have fibrils extended

- On nuclear side have nuclear cage extending into the nucleoplasm prevents chromatin from

clustering in the area and clogging pore

- Distribution of nuclear pores usually homogeneous, but in some cases clustering

demonstrated, reflecting cell structure and/or activity – usually clustering occurs when the cell is

very active in transport of mRNA and ribosomal subunits

ANNULATE LAMELLAE

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- fragments of nuclear envelope and nuclear pores floating in cytoplasm, likely bc there were too

many pores in the membrane and needed to get rid of some?

- in the cytoplasm of some germ cells (frequent in oocytes) – not common among most cell types

- found in some tumor cells

- function unknown, but demonstrate that nuclear pores can exist elsewhere than on nuclear

envelope

- these pores get disassembled by cell during mitosis just like the pores in the nuclear envelope

(because envelope gets degraded) same mechanism affects all the pores

- Model of Annulate Lamella

BioAP 3160 - LECTURE 6

EXCHANGE OF MATERIAL between nucleus and cytoplasm: only small molecules (ions, etc.) and small protein (up to about 45,000 daltons) can move freely across nuclear pores; passage highly regulated for larger molecules (ribosomes, for example) which cannot freely and passively diffuse in/out of nucleus (pores too small) - active process required - specific receptors

Proteins actively transported through nuclear pores: nuclear import signals on specific proteins (short peptides, 4-8 AA long); examples: SV40 T-antigen (viral protein that has to get in nucleus to be function, has specific sequence of 5AA Lys-Lys-Lys-Arg-Lys – if changed protein can’t get into nucleus), nucleoplasmin- highly specific short AA signals; w/ proper signal, see almost no nuclear proteins in cytosol- protein transport through the pores can be regulated (e.g. glucocorticoid receptor/hsp90)- karyophorins = nuclear transporter (many kinds), work closely with small, monomeric G proteins - Ran subclass of GTP proteins is involved in nuclear transport:

Ran-GAP (GTPase activating protein, GTP GDP) is only in cytosol so cytosol has mainly Ran-GDP.

Ran-GEF (Guanine exchange factor, GDP GTP) is only in nucleus so nucleus has mainly Ran-GTP

Gradient drives nuclear transport Import

o Importin binds cargo in cytoplasm and pases through nuclear poreo When reaches nucleus, Ran-GTP binds and importin releases cargoo Because Ran-GDP in cytosol doesn’t bind cargo receptor, unloading only happens

in nucleus – directionalityo Empty receptor with Ran-GTP is transported back to cytosol where it is hydrolyzed

back to Ran-GDP so it can begin next cycle Export

o Ran-GTP in nucleus promotes cargo binding to export receptor rather than dissociation

o Once export moves through pore to cytosol, its Ran-GTP is hydrolyzed to Ran-GDP causing release of cargo and Ran-GDP in cytosol.

o Export receptor is returned to the nucleus to complete cycle

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- selective transport of RNAs through nuclear pores: a role for the 5'cap of mRNAs

- area of rarefaction often present at nuclear side in correspondence with nuclear pores

FIBROUS LAMINA (Nuclear Lamina) – Lamins are attached to inner envelope membrane, most conspicuous in some cells of invertebrates, but present in almost all cells that have a nucleus- in mammals composed of three unique proteins, 60-70 kDa, called lamins - lamins make up one class of intermediate filament proteins-all 4 classes (lamins, keratins, neurofilaments, and desmin/vimentin/glial fibrillary have common alpha helical region, but lamins have small B sheet kink in that region- differently from other filament proteins, lamins contain nuclear transport signal (to get into nucleus)- assemble in a two-dimensional lattice (other proteins involved ?), very dynamic- lamins regulate dissolution of nuclear envelope in preparation for mitosis, cycle of phosphorylation/dephosphorylation - formation of tetramers of lamins A,C + membrane-bound lamin B - distributed in cytoplasm during mitosis - dephosphorylation at beginning of interphase with re-formation of nuclear envelope-phosphorylation of lamins by CDK’s causes dissolution of nuclear envelope-dephos. at beginning of interphase in new cell causes reformation of envelope

- fibrous lamina also thought to interact with chromatin, and to give shape & stability to the nuclear envelope

- possibly helps control which chromatin express when? unknown

RIBOSOMES AND PROTEIN SYNTHESIS

Protein Synthesis : Genetic code: 20 aminoacids, 61 codons (3 are stop codons : UAA, UAG, UGA)- Determination of reading frame: initiation codon (AUG = methionine), Kozak consensus sequence- AA’s are added to the carboxyl-terminal end of a growing polypeptide chain- The events in protein synthesis are catalyzed on the ribosome

The Translational Machinery : components and mechanisms.

- mRNA (need 5’ and 3’ noncoding seq. for transport out of nuc. and rec. by ribo)- aminoacyl –tRNAs (activation of AA, coupling with mRNA)- ribosome (small, large subunits, A, E, P sites)- soluble protein factors (initiation, elongation, termination)

Aminoacyl-tRNA's - Aminoacyl-tRNA Synthetases

- Aminoacyl-tRNA's perform two functions :

1) they activate the amino acid for peptide bond formation; anticodon somewhere in middle

2) the tRNA portion acts as an "adaptor" between the mRNA and the amino acid on the ribosome.

- Specificity of the machinery :

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a) tRNAs ; have at least one tRNA for each AA, must add correct AA to tRNA with proper anti-codon

b) aminoacyl-tRNA synthetases: have at least one aa-tRNA synth for each aa-tRNA couple,bond between 3’ end of tRNA and AA is high energy, its cleavage provides energy for peptide bond formation

Synthesis of aminoacyl-tRNAs : in two steps (specific enzymes !)

AA + ATP = adenylated amino acid attach ATP to AA

Adenylated AA + tRNA = aminoacyl-tRNA + AMP use ATP to attach aaAA to tRNA

Ribosomes : similar in prokaryotes (70S) and eukaryotes (80S)

- In mitochondria and chloroplasts (and in prokaryotic cells) present as 70S ribosomes, dissociating into 30S + 50S subunits.

- 80S ribosomes sensitive to cyclohexamide, 70S ribosomes to chloramphenicol.

- Small + large subunits assembled only during the initiation process after an mRNA has bound to small subunit

- Distinct grooves for nascent polypeptide (30 AAs) and for mRNA (35 nucleotides); P Site (peptidyl-tRNA binding site) and A site (aminoacyl-tRNA binding site)

Protein factors : Most of them act while bound to the ribosome: initiation, elongation, and termination factors.

Steps in protein synthesis:

Initiation (Eukaryotes) :1. Dissociation of 80S ribosomes into subunits;2. Activated Met-tRNAi (initiator tRNA) binds to 40s subunit, bound to P-site on small subunit;

eIF-2 required for proper positioning (rate controlling factor in some cells); formed stable ternary complex with eIF-2 and GTP (which is not hydrolyzed)

3. mRNA binds to 40S subunit - promoted by various factors - ATP is hydrolyzed - Result: 40S pre-initiation complex. 5' cap on mRNA directs binding of small ribosomal subunit to 5' end of mRNA - subunit then moves along mRNA in search of first AUG start codon

4. 60S ribosomal subunit binds to 40S pre-initiation complex; GTP hydrolyzed, factors previously bound are released.

Elongation - divided into 3 phases :1. binding of aminoacyl-tRNA to "A" site2. peptide bond formation3. translocation- ribo and mRNA move with respect to each other

Reactions occur on the surface of the ribosome and involve A and P sites catalyzed by Peptidyl transferase (a structural part of the large ribosomal subunit)

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Termination - Process begins with peptidyl-tRNA in the P site, and one of the three nonsense codons (UAA,UAG, UGA) in the A site. This configuration is recognized by appropriate release factor (RF) which binds to the ribosome (process requires GTP in eukaryotes). Presence of RF activates peptidyltransferase center, which transfers peptidyl moiety to water COOH. The various macromolecules then dissociate from the ribosome.

Polysomes : several ribosomes bound to the same mRNA molecule - common feature

Proofreading processes

- recognition of incorrectly loaded aminoacids by aminoacyl-tRNA synthetases (2 active sites on each enzyme) incorrectly paired tRNA’s preferentially dissociate

- EF-Tu bound to tRNA, after the initial codon recognition, hydrolyzes its bound GTP, before dissociating from the ribosome leaving the tRNA in place-Kinetic delay: if it’s the wrong tRNA, the hydrolysis of GTP takes long enough that ribosome has time to recognize wrong tRNA and dissociate it- Delay between codon-anticodon pairing and polypeptide chain elongation allows time for dissociation of incorrect tRNA's

Regulation of protein synthesis

- Important to determine proteins synthesized by specific cells at different stages of development/ differentiation, in response to hormones and other factors, etc.

- Several mechanisms and factors involved described, but details often still unclear.- Phosphorylation of protein factors (eIF-2), and of 40S subunit (by cAMP-dependent protein

kinase); - polyamines: affect rate and selectivity of mRNA- Control in reticulocytes by heme eif-2 phosphorylation- Virus-induced shut-off of synthesis of host proteins

o Virus-induced modification of initiation factors or altered requirement for the factors;o Virus-induced changes in intracellular ionic compositiono Ribosome modifications - Virus makes ribosome’s select only viral mRNA and not

intrageneous mRNA host genes are not expressed

Many useful antibiotics are inhibitors of procaryotic protein synthesis-some antib’s that work on euk and prok cells can be used in humans IF human cell membranes are impermeable to the drugs

BioAP 3160 - LECTURES 7-12

THE COMPARTMENTALIZATION OF CELLS - The cytosolic compartment

- Major intracellular compartments in a typical cell defined by different membrane-bounded

organelles

- The cytosol - about half of the total cell volume - organized by filaments (cytoskeleton)

- Rapid destruction of selected cytosolic proteins via the ubiquitin-dependent pathway

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Cytoplasmic proteins have highly variable lifetimes – why? UBIQUITIN

Proteins that need to be degraded by proteosomes in the cytosol are tagged with many

Ubi molecules

Process involves ubiquitin-activating enzyme that binds ubiquitin to ubiquitin ligase

Some proteins have degradation signal - sequences that express they’re ready to be

degraded, usually in N terminal.

The Ubi-ligase complex recognizes this signal and transfers ubiquitin to the protein to be

degraded. Multiple ubiquitins added chain

Proteosome is attracted and degrades protein

Heat-shock and stress-response proteins and their role in protein folding (molecular chaperones)

Chaperones bind unfolded or attempting to fold proteins and prevent them from folding

improperly or prematurely, and then help them to fold when time is appropriate

- N terminal region can fold before carboxyl has finished being synthesized, but to have

proper folding need synthesis of both ends first so can have N-C interactions

- Chaperones associate with proteins being synthesized and keeps them unfolded to give

them time to be completed before it folds

- Requires ATP to assoc. and dissoc.

- Hsp70 most common chaperone in cytosol – called heat shock because defense

mechanism to remove improperly folded proteins that have been synthesized because of

poisoning, etc.

- Hsp 60 and 10 in mitochondria

- BiP in ER lumen

STRUCTURE AND FUNCTION OF BIOLOGICAL MEMBRANES

- Selective permeability barriers – allows cell to maintain difference in inner/outer environment

- Site of selective transport systems

- Support for catalytically active molecules, and for protein synthesis (intracellular) (ER)

- Part of energy transducing systems (mitochondria)

- Conduction of electrical impulses (in nerve and muscle);

- Insulator function (myelin sheath);

- Site of specific adhesion and attachment cell components;

- Site of cellular components involved in cell recognition;

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- Site of hormone receptors, and for the transfer of their signals inside the cell;

- Intercellular communication;

- Also, ER and mito membranes have 7 times the SA of plasma membrane

- In highly active cell, more ER membrane will be present

Methods used to study membrane structure

1. Ultrastructural and morphological examination : TEM, SEM, freeze-fracture replica technique

2. Subcellular fractionation and isolation of specific membrane fractions

3. Biophysical techniques : determination of "Transition temperature", Electron Spin Resonance,

Nuclear Magnetic Resonance, etc.

4. Biochemical analysis of membranes and membrane components : analysis of lipids and

proteins, phospholipase digestion, etc.

MEMBRANE COMPONENTS : LIPIDS

- Phosphatidyl -choline, -inositol, -ethanolamine, -serine

- Derivatives of sphingosine : sphingolipids, glycolipids

- Cholesterol : regulates fluidity, confers mechanical stability

All AMPHIPATHIC : have polar head groups, hydrophobic tails

- Important features of phospholipids : different lengths of fatty acids and presence of unsaturated

cis-Double bonds (kinks - differences in fluidity)

- Phospholipids spontaneously form bilayers - hide nonpolar regions from water - High mobility on

plane of bilayer but flip-flop very rare; Can be reconstituted into artificial vesicles (liposomes)

- Variability in lipid composition of membranes

- Glycolipids carry sugar chains with sometimes very complex structures (blood groups, GM1-

receptor for cholera toxin, other cell-cell communication functions? receptors for hormones ?)

-Lipid rafts: clumps of lipids within “sea” of intermembrane space, contain high conc. of acylated

and GPI anchored proteins, present in apical polarized membrane of epithelial cells

MEMBRANE COMPONENTS : PROTEINS

- Variability in protein content of membranes - Less than 25% in myelin, up to 75% in some highly

specialized membranes, about 50% of the mass in most membranes – RBC’s are the only type

with spectrin cytoskeleton, most cells have myelin.

- Lipid bilayer serves as a solvent for membrane proteins - specific interactions between certain

lipids and membrane proteins

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- Peripheral vs integral membrane proteins - Amphipathic nature of integral membrane proteins

due to the presence of regions interacting with lipids (either spanning the bilayer or immersed

only in half of it - membrane anchors)

Current model of membrane structure : the FLUID-MOSAIC MODEL :

- Emphasis of this model is on the great degree of fluidity of the proteins in membranes. Integral

membrane proteins embedded (spanning) the phospholipid bilayer, and relatively free to move in

the plane of the bilayer

- Things can move in plane of bilayer, but are sometimes anchored to cytoskeleton!

- Flipping back and forth is highly regulated by flippases and scramblases

- Peripheral proteins not directly associated with the membrane, but with integral proteins

- ASYMMETRY OF MEMBRANES : sugar residues only on outside (surface membrane) or

inside intracellular membranes; also demonstrated asymmetry in lipids composition - Interactions

of membrane proteins with the cytoskeleton

- once inserted, lipids and proteins have polarities within their leaflet or across bilayer

ENDOMEMBRANE (EM) SYSTEM – communication between membrane systems

-Also called the Secretory Pathway

-Members of the EM system are the cell membrane, secretory vesicles, lysosomes, early and late

endosomes, the Golgi, and the ER

- ER has proteins inserted co-translationally

-Have forward (sec) and reverse (recycling) pathways which are both always functioning

-Nuclear membrane, mitochondria, peroxisome, and plastids: NOT members of EM system AND

all have their proteins inserted post-translationally

-In vesicular transport, membrane components of donor compartment become components of

target compartment

THE ENDOPLASMIC RETICULUM

Many vital biochemical processes take place in or on membrane surfaces. Internal membranes

divide the cell into specialized compartments. In most cells, particularly those active in protein

secretion (secretory cells) because secretory proteins are made on the RER, the Endoplasmic

Reticulum represents a major portion of the total cell membranes.

- ER studied by both TEM and subcellular fractionation techniques. Results obtained sometimes

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conflicting

- For reasons which will become obvious later, the ER has been divided into Rough (RER) and

Smooth (SER) Endoplasmic Reticulum : their membranes are almost identical in composition,

but have quite distinct functions

RER

- Described originally as a set of filamentous structures, which stained intensely with basic dyes

(basophilic) by light microscopy. Called Ergastoplasm (term now rarely used)

- TEM observations demonstrated the presence in cytoplasm of membrane-limited cisternae,

studded with ribosomes only at their cytoplasmic surface. The ribosomes make the RER

basophilic (RNA) and represent the main difference between RER and SER

- RER very abundant in secretory cells (secretory proteins are synthesized on RER) - both

endocrine and exocrine cells usually have large amounts of RER

- Estimates of the total surface area occupied by the membranes of the RER is up to 8,000

um/cell (guinea pig pancreatic acinar cells), representing 2/3 of the total area of membranes in

the cell

- 3D configuration of RER : fenestrated cisternae, anastomosing tubuli, isolated vesicles;

cisternae may be arranged in parallel stacks 2-4 um long, or may form extensive concentric

systems; they enclose the lumen of the RER

- Asymmetric distribution of ER enzymes - important ! membrane proteins (and also lipids) are

very asymmetric gives ER its distinct fxn

- RER cisternae continuous with outer nuclear membrane

-RER lumen continuous with intermembrane space of nuclear envelope

- Purification of subcellular fractions enriched in ER components - differential centrifugation –

useful for separating RER and SER and ribosome’s from RER because of their different densities

- Fractions rich in 3 ribosomes originally called "Microsomes"; correlated biochemical and

ultrastructural studies established the identity of microsomes and endoplasmic reticulum

(microsomes are obtained as an artifact of subcellular fractionation by fragmentation of the ER

during Homogenization and resealing of the fragments into vesicles)

-RER makes all the secretory proteins of the cell and all the EM system members’ membrane

proteins

-Also the site of the first step of glycosylation of glycoproteins

SER

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- Set of membrane-limited tubules - continuous with RER but devoid of ribosome

-Tubules looks a lot like vesicles in cross section, hard to distinguish the two

- Well developed in some cells, but difficult to identify by TEM in many cell types

- Biochemical properties of RER and SER, apart from the disparity in ribosomes content, very

similar. SER and RER have all the same enzymes, localized similarly

- Highly versatile organelle very well developed in cells involved in active metabolism of lipids or

metabolism of xenobiotics (induction by phenobarbital in liver)

- Steroid secreting endocrine glands: often present as extensive spiral or concentric arrays of

fenestrated cisternae organized around lipid droplets – SER produces steroids

Main functions of RER

- Synthesis of membrane proteins and secretory proteins (translocated into the lumen of the

RER as they are synthesized by membrane bound ribosomes)

- glycoprotein synthesis – first step of glycosylation of glycoproteins

Main functions of SER

- Synthesis of lipids (cholesterol, phospholipids, triglycerides, steroids) – SER enlarged in steroid

producing cells. All membrane lipids of cell originate in SER

- degradation of glycogen

- detoxification processes (drugs, steroid hormones, carcinogens, etc) – SER modifies lipophyllic

wastes that are hard to enter bloodstream so they are water soluble.

- transport of ingested lipids inside intestinal cells

-Ca++ sequestering and release (important in signal transduction pathways)

-Phospholipid synthesis always begins in the cytoplasmic leaflet of the ER membrane

- Starts with fatty acyl CoA and glycerol 3P (acyl transferase) phosphatidic acid

(phosphatase) diacylglyercol (choline phosphotransferase) phosphatidyl choline

phospholipid added to cytosol half of lipid bilayer

- If outer leaflet is always growing, how do we get the two to even out? TRANSLOCATORS

-Translocators explain the symmetry of the overall NUMBER of lipids in each layer and the

asymmetry of the TYPES of lipids in each layer

THE SIGNAL HYPOTHESIS (universally accepted)

-Explains the association of specific ribosomes and their attached mRNA’s with ER membrane –

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cotranslational import into lumen of ER

1. Secretory and membrane proteins are made by ribosome’s bound to ER – ribosomes only

attach when synthesizing

2. Free and bound ribosome’s can exchange subunits – no difference

3. Free and bound ribosome’s are identical – what is different is product of translation = signal

4. Products of protein synthesis in RER membrane are always segregated in a compartment,

never in the cytosol (ER, Golgi cisternae, Golgi vesicles, out of cell)

5. During or shortly after translation in RER membrane, certain modifications can be made in the

ER lumen.

6. Signal hypothesis: information determining the association of certain ribosome’s with the RER

membrane is contained in the N-terminal segment of the protein – the “signal peptide”

- This is all true, but not totally complete because now know that signal is sometimes internal and

not N-terminal

HOW SIGNAL PEPTIDES ARE UTILIZED

-Secretory protein synthesis always begins at a free ribosome in the cytosol

-About 25-30 AA into synthesis, the signal peptide is revealed (previously hidden by large

subunit)

- The SP is recognized by the signal recognition particle (SRP) which is made of protein and RNA

- Binding of the SRP temporarily halts translation and gives the free ribosome time to move to the

ER membrane and find an SRP receptor and a translocator

- The SRP receptor recognizes the SP-SRP-ribosome complex and binds the ribosome

- The SRP receptor binding the ribosome helps to guide the peptide to a channel in ER

-once the polypeptide links the ribosome to the ER , the SRP-SRP receptor dissociates

- translation continues until peptide fully synthesized and signal peptidase cleaves SP, which then

remains in ER membrane

-SP either gets degraded in ER membrane OR if the SP is not completely, can become

membrane bound part of ER membrane protein

ER MEMBRANE PROTEINS: SINGLE AND DOUBLE PASS

-Single pass (class 1) proteins made by:

start transfer sequence (SP) and stop transfer sequence (stops protein from entering

lumen of ER)

translation continues until stop transfer sequence is reached

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C-term of peptide sticks out into cytosol

Start transfer sequence is cleaved and N-term of peptide sticks into ER lumen

Can become anchored in GPI-anchor of lipid raft

-Double pass proteins made by: multiple start and stop transfer sequences. Same as single

except start transfer sequence never cleaved and now protein has 2 transmembrane segments

ALL PROTEIN SYNTHESIS STARTS ON FREE RIBOSOMES!!

PROTEIN ASSEMBLY & DEGRADATION IN THE ER

- Translocated polypeptide chains fold in the lumen of the ER

- As peptide is synthesized, it is bound by chaperones in the ER lumen to prevent folding before

the entire thing is synth. then they help it fold once it’s done synth.

- Protein disulfide isomerase and its role in correct disulfide bridges formation

-PDI is ONLY found in the lumen of the ER all disulfide bridges are formed in lumen of ER

- makes sense because usually only secreted proteins have disulfide bridges and all secreted

proteins are made in ER!

- Peptidyl prolyl cis-trans isomerases: abundant and widely distributed - inhibited by

immunosuppressive agents

-PctI catalyzes switching between cis and trans peptide bonds in proline residues

- Role of binding proteins-chaperones (BiP, stress-response proteins)

-BiP role: binds proteins as they enter the lumen and prevents improper folding, hydrolyzes ATP

to provide energy needed to pull protein across ER membrane

- Also makes sure Ig have heavy AND light chains before they can be sent out of ER

-Calnexin and calreticulin: chaperone proteins that require 1glucose on protein to be able to bind

- the longer a protein is in the ER, the more glucoses it loses

- if it’s still unfolded AND loses all glucoses, it must be degraded bc will never fold right

- if it’s still unfolded AND glycosyl transferase keeps adding a glucose back on, calnexin and

calreticulin will keep binding it and helping it fold

- Role of ATP binding and hydrolysis by stress-70 proteins: provides energy needed to pull

peptide across membrane

- Selective protein degradation: if the protein still isn’t folded right, it is recognized by reverse

translocators (Sec61 complex) that bring it out into cytoplasm where it can receive a multi-Ubi tag

and be degraded by proteosome

- Glycosylation of proteins in ER: N-linked oligosaccharides added in lumen of ER to Asn (N)

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residue on protein being translocated

- The N-linked oligosaccharide is built sugar by sugar in association with dolichol (lipid) and then

whole thing added (en bloc) to protein

TRANSFER of PROTEINS and LIPIDS from RER to cis-GOLGI

- takes place via vesicles, budding from the transitional elements of the RER: these are areas of

RER where there are no ribosome and there is a lot of blobs/projections that will become vesicles

- Intermediate compartment: Also called the salvage compartment or cis-Golgi-network

- formed by fusion of vesicles that have just left ER

- between RER and Golgi, re-cycling of resident RER proteins: if something is sent out of

transitional network by mistake, the salvage compartment recognizes this and sends a vesicle

back to the ER with the molecule in it (usually resident ER proteins)

- mediated by specific receptors: these receptors are constantly cycling from ER-intm. comp.-ER

even if they don’t “catch” anything in the intm. comp.

- role of KDEL and KXXX sequences: BiP has a KDEL sequence, other resident proteins have

KXXX sequence that bind directly to COP I coats to be sent back to the ER

- ER, intm. comp. and Golgi distinguished by particular enzymes present in them

The GOLGI COMPLEX

- Observed by light microscopy (Golgi) after staining with heavy metals

- Diagram of Golgi apparatus: thin in central portion, expanded at periphery; Convex - forming

(Cis) face / Concave - maturing (trans) face POLARITY

- Histochemical staining of the Golgi complex: only seen after leaving prep for too long

- Trans-tubular network (visualized in liver cells after Lowycryl low temperature embedding)

Evidence for distinct subcompartments (monensin blocks cis-to-trans transfers)

-Cis Golgi network: intermediate compartment + Cis Golgi, sorting center

-Cis-Golgi : impregnated with osmium, phosphorylation oligosaccharides on lysomomal proteins,

removal of mannose

-Medial Golgi : removal of mannose (and glucose ?) from glycoproteins; addition of GlcNAc

- Trans Golgi : staining for thiamine pyrophosphatase, staining for acid phosphatase, addition of

terminal sugars to glycoproteins, proteolysis of preproteins, sulfation of glycoproteins, addition of

Gal

- Trans-Golgi-Network: some investigators consider it a separate organelle

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-True shipping and packaging center

-Much larger than rest of Golgi apparatus

Transfer of proteins and lipids between Golgi cisternae: via vesicles, non-clathrin-coated, budding

from the rim of one cisterna and fusing with the next (see next Lecture); tubular connections may

be involved in reverse transfer (transcis)

Major functions of Golgi complex

- Synthesis of glycoproteins and glycolipids

- Proteolytic processing of preproteins (Trans Golgi)

- Packaging of lipoproteins and formation of secretory granules (Trans Golgi network)

- Recycling of surface membrane components (Cis Golgi network (intm. comp.))

- Membrane biogenesis

- Control center for distribution of membrane components into different cellular membrane

fractions (Trans golgie)

GLYCOPROTEIN SYNTHESIS

N-linked vs O-linked oligosaccharide chains

- N-linked oligosaccharides: initial step during translation in the RER membrane (en bloc)

ER Lumen = initial Golgi

Golgi Lumen

o Golgi mannosidase removes three more mannoses forms high-mannose

oligosaacharide. Important for marking lysosomal enzymes. If not used for lysosme,

two more mannoses removed by medial golgi, then others added

Three glucoses removed in ER. Why add three glucoses just to remove them before it gets

out of the golgi? Because calnexin chaperone needs a glucose to chaperone.

- extensive processing later in the RER and in the distinct portions of the Golgi complex: N-linked

oligosaccharide can become complex (original oligo. is trimmed and more sugars are added) or

can become high mannose (some original oligo. trimmed but no new sugars added)

- Specific glycosidases cleave different biosynthetic intermediates

- O-linked oligosaccharides: sugars added sequentially (no initial block transfer)

Concentration of secretory products.

- Concentration is not dependent on continuous expenditure of energy.

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- Concentration (which continues during maturation of secretory granules) apparently results from

the formation of osmotically inactive aggregates: as vesicles move through Golgi network, in the

granule positive and negatively charged proteins get together and neutralize the charges. When

charges are lost, there’s a change in osmotic activity in granule water moves to cytoplasm by

osmosis. Lose some of their cytoplasmic content (sent back to Golgi in clathrin coated vesicles)

and the molecules within them form aggregates, water leaves vesicle contents become

concentrated. No net movement of water through channels.

- Constitutive secretory pathway – don’t concentrate, vesicles containing (collagen,

immunoglobulins, etc) are immediately discharged to the surface, not retained in secretory

granules. Don’t need concentration

Secretory Granule formation

Once the secretory proteins reach the trans face of the Golgi complex, they are further

concentrated and packaged in membrane-limited vesicles, vacuoles, or granules.

Temporary storage of secretory products in membrane-bound vacuoles and vesicles

Under normal, resting conditions, secretory granules appear incapable of recognizing and fusing

with each other, although in some cells they are closely opposed to each other. The reasons for

this lack of fusion are unknown at present. (picture of resting mast cell with lots of vesicles sitting

near cell membrane)

-in order for contents of vesicle to be secreted, vesicle membrane must dock on cell membrane

and fuse with it to release contents outside cell.

VESICULAR TRANSPORT

-The Constitutive secretory pathway: once a vesicle is made and sent to membrane it gets

secreted immediately

-The Regulated secretory pathway: only some cells, vesicle is not released from trans-Golgi until

cell receives signal to do so

LYSOSOMES

- Discovered by chance: irregularities in enzyme assays

- Membranous bag of hydrolytic enzymes used for controlled intracellular digestion of

macromolecules to micromolecules to be reused in the cell

About 40 enzymes known to be present in lysosomes - all are acid hydrolases (pH optimum

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about 5) - extra protection against activity if released in the cytoplasm (other protection =

membrane) has DNases, phosphatases, lipases – can digest almost everything. Enzymes

synthesized in ER – high mannose has phosphate group added in cis golgi – sorting marker so

transported to lysosome by M6P receptor. Mannose 6P blocks further processing of carb chains.

- Membrane of lysosomes contains transport protein which utilizes energy of ATP hydrolysis to

pump H+ into the lumen of lysosomes (H+ pump maintains low pH in lysosome)

Lysosomal membrane resistant to the enzymes it encloses, and impermeable to their substrates -

Process of lysosomal digestion carried out inside the lysosomes

- Histological staining for lysosomes

- Primary lysosomes often found clustered around the Golgi complex

-Lysosomal enzymes work at pH lower than cytosolic, so that’s another defense mechanism

against lyso. enzymes getting loose and destroying cell

Main role of lysosomes : cell digestive system - digestion of intra- and extracellular (lysosome

secreted to digest stuff outside cell) debris, phagocytosed organisms, cell nutrition (cholesterol

assimilation)

1) Digestion of materials taken up by endocytosis (coated pits, endosomes)

2) Breakdown of intracellular material – autophagy – during development and healing

3) Digestion of phagocytosed particles and microorganisms - only in specialized cells

4) acrosome of sperm cell contains enzymes to break down oocyte cell membrane, bone

reabsorption

End results : small MW components enter cytoplasm and are re-utilized

- lipofuscin pigments: accumulate with age, evidence of things not degraded over time

- Lysosomes involved in many human diseases and syndromes: storage diseases

- Storage diseases : accumulation within the cells of various substances (glycogen, glycolipids,

etc.) due to lack of specific lysosomal enzyme - genetic defects – too much of something kept in

cell that should have been degraded in lysosome

Synthesis of lysosomal enzymes

- Synthesized in ER and transported to the Golgi

- Move from cis to trans-Golgi like secretory proteins

- Transport vesicles that deliver lysosomal enzymes to endolysosome bud from the trans-Golgi

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network

- Selection of lysosomal enzymes due to the presence on them of a unique marker: mannose-6-

phosphate -added to N-linked oligosaccharides in the cis-Golgi

- Key enzyme : GlcNAc phosphotransferase; deficient in I-cell disease

-GlcNAc phosphotransferase functions in the cis-Golgi to add the M6P tag to the lys. enzyme

- Know to add the tag because of lysosomal “patch” on enzyme (details unknown)

- Receptor proteins cluster and become concentrated in clathrin-coated vesicles in trans-Golgi;

they bind mannose-6P at pH 7, release it at pH 6: to transport lys. enzymes, M6P receptor

proteins in trans-Golgi cluster and bind m6P at pH 7, clathrin coated vesicles form, leave trans

Golgi, lose coat, move to lysosome and fuse with it, releasing M6P tag at pH 6

- After leaving Golgi, vesicles lose clathrin coats before fusing with endosomes/endolysosomes

- Receptors recycle to trans-Golgi; not found in mature lysosomes: the receptors that brough the

enzyme to the lysosome go back to the trans-Golgi to pick up more enzyme

- Recycling inhibited by ammonia, chloroquine

- In some cells mannose-6P receptors also present on outside of the cell surface, to recover

lysosomal enzymes secreted into extracellular medium

PEROXISOMES

- NOT part of IM system, post translational import of proteins, all proteins made in cytosol

- Particles which contain most of cells' Catalase + one or more enzymes which use molecular

oxygen to remove hydrogen atoms from specific substrates (peroxidase, D-amino acid oxidase,

urate oxidase); Catalase uses hydrogen peroxide produced to oxidize a variety of substrates

(phenols, formic acid, formaldehyde, alcohol) or to produce water + O2 (safety device which

prevents accumulation of oxidizing agent, hydrogen peroxide)

- function = hydrogen peroxide detoxification, alchohol detoxification

- In liver oxidize about half of ethanol ingested

- Found in liver, kidney, protozoa, many cell types of higher plants, and other cells (usually in

lower amounts); very diverse organelles, even in different cells of a single organism may contain

different sets of enzymes

- Identified by specific staining with the diaminobenzidine reaction for peroxidase activity – look

identical to lysosomes, the only way to distinguish is that diaminobenzidine darker color

- Typical inclusion found in rat hepatic peroxisomes : the nucleoid:

- Peroxisomes until recently thought to be formed as dilations of the RER; it is now clear that new

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peroxisomes are only formed from pre-existing ones by growth + division (similar to mitochondria)

fission

- Grow by uptake of new peroxisomal proteins from cytosol

- All components of peroxisomes imported from the cytosol (amino acid sequence acts as a

specific signal); receptor proteins on membrane surface

- Zellweger syndrome: due to defect(s) in peroxisomal proteins import

EXOCYTOSIS

2 distinct steps: bilayer adherence and bilayer joining (remember 4 leaflets, 2 bilayers)

- Membrane fusion catalyzed by fusogenic proteins

Exocytosis of secretory proteins stored in granules requires:

1. secretagogues – signals, interact with receptor sites located in the plasma membrane. =

hormones, neurotransmitters (mediated by increase in calcium or cAMP)

2. ligand-receptor interaction transduced into a proximal biochemical intracellular response:

mobilization and elevation of intracellular free Ca 2+concentration, or activation of adenylate

cyclase and elevation of intracellular cyclic AMP (cAMP) levels.

- Calcium plays a critical role in regulating exocytosis in a number of systems.

- The overall process is independent of protein synthesis, but requires metabolic energy – ATP

hydrolysis

3. Movement of the secretory granule from its site of formation/storage to the plasma membrane.

-sometimes involves microtubules, inhibited by colchicines (inhibit microtubules)

4. Fusion of the secretory granules with the plasma membrane, and discharge of the granule

content regulated by specific enzymes

- Docking = vesicle comes to cell surface but still maintains membrane bilayer. Fusion =

membrane bilayer is broken and secretion occurs.

ENDOCYTOSIS

- General term applied to the uptake by cells of particles by encirclement with cell processes

(phagocytosis), fluid in vacuoles formed by invagination of the surface membrane

(pinocytosis), specific uptake of extracellular proteins/peptides/other materials (receptor-

mediated endocytosis, potocytosis)

Pinocytosis = FLUID

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- Vesicular uptake of fluid containing low MW solutes, soluble macromolecules, colloidal particles

(too small to be visualized with the light microscope), etc.

- Can be distinguished in two types :

- micropinocytosis (fluid taken up in minute invaginations of the cell surface visible only by TEM)

-caveolin coats on caveolae (tiny invagination) involved in this – cells in culture use this to take

up culture with nutrient in it, extensive form of feeding.

- macropinocytosis (undulating folds capture droplets visible with the phase-contrast microscope -

typically occurs at the thin peripheral portion of flattened cells in culture to eat) (even less specific

than micro in that no known proteins are needed for this) Lamellipodia (membrane foldings)

-Non specific in that you do not need certain proteins to be able to pinocytize and you grab

whatever you grab – involves folding of plasma membrane on itself.

Receptor-mediated endocytosis

- This process is highly specific (requires ligand-receptor interaction) and involves many

physiologically important molecules, happens in most tissues/cells

- Specialized regions of the surface membrane involved : clathrin coated pits - following

endocytosis, coated vesicles formed

- Coat composed primarily of a single protein, clathrin (MW 180,000) - minor proteins also

present specifically associated with clathrin (adaptins, etc. - see next Lecture)

- Polygonal network of clathrin, visualized by TEM, SEM (triskelions)

- Uncoating ATPase (member of hsp70 family of stress response proteins) removes coat from

clathrin-coated vesicles as soon as it enters cell

- There are also other 'coated vesicles' in cells (not coated with clathrin) involved in intracellular

vesicular transport processes (e.g. among Golgi cisternae - see next Lecture)

- Role of receptor-mediated endocytosis in cholesterol import - LDL receptors: LDL receptor +

LDL brought into cell and separated in lysosome, LDL released into cytosol and receptor sent

back to cell surface

- Endocytic vesicles deliver their contents to endosomes (early and then late endosomes) which,

after fusion with Golgi derived transport vesicles containing lysosomal enzymes, become

endolysosomes, and finally lysosomes

- Early endosomes are important sorting centers, decide some things should cycle back to cell

surface but other components should move on.

- Potocytosis: sequestration and transport of small molecules by caveolae. Specific receptors

involved. Don’t generate vesicles, have envaginations forming that are closed to outside then

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reopen. Best studied example: the folate receptor

-Don’t need a folate gradient to bring folate in, will still transport it in

-Folate receptor on cell surface binds folate (LIGAND), caveolin causes tiny invagination

(caveolae) and folate is released into cell

-no “vesicle” actually enters cell. The receptors concentrate folate in these little invaginations

so when the channel opens you have more folate there then in cytoplasm so it moves down it’s

concentration gradient and into the cell.

- Transcytosis (transfer from one extracellular space to another in polarized epithelial cells)

- IgG uptake in newborn small intestine

- IgA secretion in epithelial cells

Phagocytosis = LARGE PARTICLES (bacteria)

Ingestion of particles large enough to be visible with the light microscope. In mammals

macrophages and neutrophils very active in phagocytosis.

1) Attachment by interaction of a ligand on the particle with surface receptors on the membrane of

the phagocyte;

2) Ingestion : zipper-like process

- Attachment does not require energy, ingestion does and is temperature-dependent

- Large, flat clathrin patches, similar to those characteristic of coated pits and vesicles (see

receptor-mediated endocytosis) observed on the cytoplasmic side of phagosomes;

- Following internalization, phagosomes fuse with lysosomes

VESICULAR TRANSPORT: SUMMARY

- Signal peptides and signal patches - mediate at least some of the intracellular sorting

processes

1. import into nucleus (e.g. large T, nucleoplasmin) nuclear import signals

2. import into ER (signal peptides) Signal Peptide, start and stop transfer signals

3. retention in lumen of ER (KDEL) KDEL=BiP and KXXX=others, resident ER prot

4. import into mitochondria specific import signals (post-transl)

5. import into lysosomes M6P tag, receptor proteins, etc.

6. endocytosis ligands, clathrin, other signals

-early endosome = sorting center, can move stuff back to cell surface, to late endosome, or

can become late endosome

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- Constitutive vs Regulated secretory pathways – SPECIFICITY OF COMPARTMENTS

- different types of coated membrane regions/vesicles (clathrin, coatomer, caveolin)

Clathrin = endocytotic vesicles and for vesicles that bud from trans golgi to generate vesicles

filled with lysosomal enzymes that fuse with lysosomes. Same clatrhin, different linker proteins

COP I = forward pathway from cis golgi trans and then plasma membrane & reverse pathway

golgi ER Cop II = ER cis golgi network

- assembly & disassembly of clathrin coats, adaptins: same clathrin throughout, uses different

adaptins in different vesicles depending what it’s transporting and where it’s coming from

- coatomer-coated vesicles & their role in intracellular vesicular transport (ER-to-Golgi; cis-to-

trans Golgi; TGN-to-surface membrane); 7 subunits (COPs), ARF, monomeric & trimeric G

proteins

- ARF (monomeric GTPase, fatty acid tail) regulates both assembly & disassembly of coatomer

coats assembly: exchange GDP/GTP (catalyzed by GNRP, inhibited by Brefelden A), fatty acid

tail exposed, insertion into donor membrane, recruitment of coatomer subunits, budding, pinching

off of vesicles disassembly: docking at target membrane, GTPase-activating-protein, GTP

hydrolysis, ARF retracts tail, disassembly of coat GTP = tail, GDP = no tail (can’t bind)

- v-SNAREs, t-SNAREs & Rab proteins determine selectivity of vesicles docking

-proteins on vesicle surface recognized by complementary proteins on target compartment

surface

- Rab proteins: GTPases whose affinities for different ligands change when bound to GDP or

GTP

Donor Compartment

o Coat helps select what is incorporated in a certain vesicle and what is not.

o Formation of bud involves aggregation of Rab-GTP that’s specific for donor

compartment. GTP is essential for binding of Rab to vesicle, neded for uncoiling of

lipophyllic tail.

Binding to target compartment

o Membrane fusion, Rab effector binds to Rab, GTP hydrolyzed and Rab becomes

soluble not membrane bound – donor compartment has right GEF to make

membrane soluble again (attach GTP cause exposure of lipophyillic tail)

-when Rab-GTP is bound to vesicle it has a lipid tail which is recognized by Rab effector on

target membrane (in addition to v-t SNARE interaction)

- after fusion, Ran-GTP is hydrolyzed to remove from membrane (now part of target

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membrane) and sent back to donor compartment (no more lipid tail)

-GDP is replaced by GTP by GEF and Ran-GTP can be used again in another vesicle

- fusion of vesicles to target membrane:

-content of vesicle becomes part of luminal comp. of target and vesicle membrane components

become part of target membrane

-complex process: NSF: cytosolic component that aids in dissociation of v-SNARE and t-

SNARE (necessary to complete fusion) by hydrolyzing ATP and providing energy for dissoc!

- SNAPs involved

CELL SURFACE POLARITY

- cell surface domains (e.g. apical/basolateral)

- Cellular junctions are generally assumed to function as barriers

-Tight junction prevents proteins from moving laterally from domain to domain

- GPI-anchored proteins are found exclusively in the apical plasma membrane

Cell surface polarity in Epithelia

- Epithelial cells are organized into sheets that separate compartments of the organism. The cells

in the epithelium are linked through junctional complexes so that they form a selective

permeability barrier. Epithelial cells are specialized to perform a wide variety of vectorial

functions. Transporting epithelia, such as those of the renal tubule, absorptive epithelia such as

those of the intestine, and secretory epithelial cells (e.g. hepatocytes) are typical examples of

epithelia that create and maintain concentration gradients between the compartments they

separate. These vectorial functions reflect, and depend on, the polar organization of the epithelial

cells. Epithelial cells accomplish these functions by localizing distinct sets of cell surface

components to separate regions of the plasma membrane. The basal aspect of the epithelial cell

layer usually rests on an extracellular matrix, which is often organized into a basement

membrane.

Apical and basolateral domains differ in both lipid and protein composition. Lipids studied in

only a few cell types : in the absorptive villus cells, glycolipids and cholesterol are much more

abundant in the apical surface, phosphatidylcholine in the basolateral domain.

Proteins studied in much more detail, and represent typical markers of epithelial cell polarity.

For example, sucrase, aminopeptidase, lactase are only in the apical surface of the intestinal

villus cells, Na+, K+

-ATPase in the basolateral membrane.

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-Golgi uses different types of vesicles to get stuff to apical and basolateral membranes, mistakes

in this are fixed by endosomes

Sorting at level of transgolgi network, some buds are destined for apical or lateral/basal

membrane.

Components are moved to lateral membrane, some stay because they belong there,

others are retaken up by endocytosis and end up in early endosomes. Early endosomes

are sorting centers and move back some component to lateral/basal membrane because

they belong there but move some up to apical.

Sorting done in endosome after insertion in lateral membrane.

Use of Viruses in the study of epithelial cell polarity : Rodriguex-Boulan and Sabatini were

the first to demonstrate that enveloped viruses bud in a polarized manner from infected MDCK

cells. This finding paved the way for the use of viral glycoproteins as probes to study the

biogenesis and transport of apical and basolateral proteins in epithelial cells in culture. Following

infection by enveloped viruses, host protein synthesis is suppressed and large quantities of viral

surface glycoproteins are synthesized. This amplification facilitates the studies of plasma

membrane biogenesis; the assumption is made that in infected cells the basic processes of

membrane protein synthesis and intracellular transport are not altered. In the case of MDCK cells,

it was shown that the G protein of vesicular stomatitis virus (VSV) is inserted mainly into the

basolateral plasma membrane, whereas the spike glycoproteins of influenza virus behave as

apical plasmalemmal proteins.

The Sorting process of newly synthesized membrane proteins destined to different parts of the

surface membrane is essentially still a mystery. Apical and basolateral proteins seem to remain

together during transport through the Golgi complex; The trans-most part of the Golgi complex is

emerging as a good candidate for the sorting process, and the recently described Trans-tubular

network continuous with the Golgi apparatus may be an important part of the sorting machinery.

BioAP 3160 - LECTURE 13

Structure of Mitochondria

- Mitochondrion bounded by smooth-contoured outer membrane (about 7 nm thick) and an inner

membrane ; two membranes separated by a space (membrane space or intracristal space)

- Mitochondrial Matrix inside, gel-like.

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- Matrix granules: about spherical, osmiophilic inclusions. DNA, bound Ca++, ribosomes

- Ribosomes and DNA inside the mitochondria.

- Number of mitochondria/cell and number of cristae/mitochondrion related to energy requirement

- Cristae very variable in shape - sharp angulations, slender villi, tubular cristae (steroid secreting

cells), branching tubules (protozoan), prismatic tubules (triangular in cross section).

- Crystalline and other inclusions frequently observed.

- Number of mitochondria/cell very variable: liver, 800-2000; oocytes of echinoderms, 150,000;

Giant amoeba Chaos chaos, about half million. May be randomly dispersed in cytoplasm, or

concentrated around the cell center.

-Mitochondria are very motile within the cell and usually know where to go (where ATP needed) –

in kidney epithelium, lots of Na/K pumps there so need a lot of ATP

- Mitochondria pick up a lot of calcium (calcium homeostasis – different than ER, calcium taken

up isn’t easy to release). Apoptosis starts with release of cytochrome C in mitochondria

Main functions of mitochondrial "sub-compartments"

Matrix: - oxidation of pyruvate, fatty acids

- citric acid cycle

- DNA replication, protein synthesis (mitochondrial tRNAs, ribosomes)

Inner membrane : VERY highly selective

- respiratory chain

- ATP synthesis (ATP synthetase)

- specific carriers

Intermembrane space: - phosphorylation of nucleotides other than ATP

Outer membrane: - permeable to molecules less than 10,000 daltons (porin)

Isolation and characterization of Mitochondrial membranes.

- Lipid content: outer, 40%, inner, 20% - outer membrane contains more cholesterol, higher in

phosphatidyl inositol, lower in cardiolipin.

-Inner membrane has a LOT of cardiolipin: a lipid that makes the inner membrane super selective

- Mitochondrial enzymes are highly compartmentalized.

Mitochondrial DNA

- Localized in the matrix (multiple copies) - Typical properties:

1. Circular molecule, 5-6 µm long, 15-17 kb pairs (in animals)

2. Buoyant density in CsCl differs from that of nuclear DNA

3. No histones

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4. Codes for two rRNA's, set of 22 tRNA's (all mitochondria specific), 13 protein-coding

sequences (ATPase, Cytochrome b complex, cytochrome oxidase complex)

5 . Little space for regulatory DNA sequences, different genetic code genes tightly packed!

6. All other mitochondrial proteins coded for by nuclear DNA.

- Mitochondrial ribosomes: smaller than cytoplasmic ribosomes, like bacterial ones

- Symbiont hypothesis: Mitochondria and Chloroplasts are intracellular prokaryotic parasites?

- Mitochondrial genes are an example of non-Mendelian inheritance, and are maternally inherited

-Mat. inherited bc majority of cytoplasm in a zygote comes from egg, and mitochondrial DNA is

floating in cytoplasm zygotes get mostly maternal mitochondrial material

Synthesis of Mitochondrial Proteins and lipids

- Lipid components of mitochondrial membranes imported from ER via carrier proteins

- Most of the proteins found in mitochondria are coded for in the cell nucleus, synthesized on free

ribosomes, and imported into the mitochondrion by specific processes. post-translationally

4 distinct destinations : outer membrane, intermembrane space, inner membrane, matrix space

Translocation of Matrix proteins

- proteins imported from cytosol within few minutes from completion of synthesis

-usually bound by chaperones once they’re synth. so they don’t fold before they get where they’re

going in the mitochondria. Precursor proteins for the mitochondria have a signal sequence,

mitochondrial import sequence that is recognized by TOM complex. Difference b/w matrix protein

and inner membrane protein is existence of second signal sequence that directs it through the

inner membrane.

- they have almost always a signal peptide (minimum about 12 amino acids)removed by a signal

peptidase in matrix

- receptor proteins for signal peptide in outer mitochondrial membrane ?

- import specifically at contact sites between outer and inner membranes

- two main translocators: TOM and TIM (2 types of TIM)

- energy required for import: 2 steps

1. TOM: initial penetration of signal peptide: occurs at low temperature, requires ATP hydrolysis

2. TIM: movement of remaining protein chains into matrix: requires electrochemical gradient

across mitochondrial inner membrane

- unfolding of proteins for translocation; chaperones (members of hsp60 family) and ATP required

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Protein transport into intermembrane space and inner membrane

- First proteins transported into matrix and first signal sequence is cleaved

- then second, very hydrophobic amino acid sequence, uncovered by cleavage of signal peptide,

acts as a signal for reinsertion (“export”) into inner membrane

- inner membrane proteins remain inserted into membrane, without cleavage of the second signal

sequence

- if destined for intermembrane space, steps above are followed, ending with the cleavage of the

second signal sequence so that protein is free in intermembrane space

Insertion of cytosolic proteins into outer membrane

- ATP hydrolysis needed, but not membrane potential - mechanism still unclear

Division of mitochondria

1. DNA replication - synthesis of mitochondrial ribosomes and structural proteins?

2. Pair of membranes forms septum extending across the organelle. Distinctive structure, not

just an extension of the cristae.

3. Circumferential fold of outer membrane invades the space between septal membranes.

4. When advancing edges of fold meet and fuse, separation of daughter mitochondria is

completed.

MITOCHONDRIAL BIOENERGETICS - CHEMIOSMOTIC THEORY

- Why is ATP an energy source? Equilibrium ATP ADP + Pi

- ATP concentration in the cytosol is kept much higher than at equilibrium; the tendency of ATP

to reach equilibrium, that is, the large decrease in free energy associated with ATP hydrolysis

under the conditions normally present inside living cells, is what drives coupled, energetically

unfavorable reactions – ATP has a tendency to hydrolyze, that tendency drives rxns.

- Mitochondria and Chloroplasts are primarily responsible for the rapid conversion of ADP into

ATP, which maintains the cytosolic ATP concentration much higher than at equilibrium.

- Glycolysis (substrate level oxidation) also produces ATP while converting glucose into pyruvate

in the cytosol, but its ATP yield/glucose is much lower than for oxidative phosphorylation.

Inside Mitochondria (in Matrix) oxidative metabolism fueled largely by fatty acid (derived from

the storage form, fat), and pyruvate (produced from glucose, by glycolysis in cytosol). Selectively

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transported into mitochondria.

- fatty acids broken down in the fatty acid oxidation cycle to Acetyl-CoA

- pyruvate also converted to Acetyl-CoA

- Acetyl-CoA enters Krebs cycle and generates NADH, FADH2 which then interact with the

respiratory chain

CHEMIOSMOTIC THEORY

- Mitochondrial inner membrane provides a framework for e- transport processes that convert

energy of NADH or FADH2 into ATP.

- Chemiosmosis - link between chemical and transport processes - e- are passed along the

electron transport chain of proteins, embedded in an ion impermeable membrane, and the energy

released in the e- passage is used to pump protons form one side of the membrane (the matrix

side) to the other (the intermembrane space). pH gradient is generated (>7 in matrix, <7 in the

intermembrane space) and membrane potential (negative in matrix, positive in intermembrane

space).

- This generates an Electrochemical Proton Gradient with a corresponding Proton Motive

Force -In mitochondria of a typical cell, Na+/H+ exchange maintains pH in matrix relatively close

to neutrality (bring Na+ into matrix to keep pH from getting too high w/ loss of H+)

- Proton Motive Force, in addition to driving ATP synthesis, also drives various transport

processes (PO4 influx, Ca2+ influx, ATP/ADP antiport system –ATP is sent out of

mitochondria into cell against its gradient) against the corresponding concentration gradients.

ATP-Synthetase (F1-Fo Complex) - couples H+ flux inward (driven by Proton Motive Force) to

ATP synthesis (from ADP + Pi). Large complex (9 polypeptides) acts as a transmembrane H+

channel.

- Purified ATP synthetase (without membrane) actually is an ATPase: hydrolyses ATP

-reverse function stimulated when cell senses that there’s too much ATP

- Inner mitochondrial membranes, without F1 fragment of ATP synthetase, oxidize NADH in the

presence of O2, but no ATP is synthesized in the process (the portion of ATP-synthetase

embedded in the membrane, the Fo fragment, makes membrane permeable to H+ and effectively

dissipates the proton gradient).

- F1 fragment, added back to stripped inner mitochondrial membranes, reconstitutes ATP

synthesis.

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- Elegant "demonstration" of Chemiosmotic theory: artificial coupling, in synthetic liposomes

- Under special conditions, ATP synthetase can be made to function in reverse: ATP is

hydrolyzed with concomitant transport of H+ in reverse.

- Balance between free-energy change for moving 2-3 H+ across membrane (proportional to

protonmotive force) and free-energy change for ATP synthesis in matrix.

- When ATP hydrolyzed in cytoplasm, ratio ATP:ADP in matrix falls, and ATP synthetase starts

again to make ATP. (when cell senses too little ATP in cell, starts to make more ATP)

- H+

IONOPHORES uncouple oxidative phosphorylation by dissipating H+ gradient

- Brown Fat Cells energy of oxidation entirely dissipated as heat: hibernating animals, human

babies. (H+ gradient uncoupled from ATP synthesis so H+ just diffuses back down its gradient

and heat is released, but no ATP made)

RESPIRATORY CHAIN

Overall reaction: H2+1/2 O2 = H2O carried out in several steps. H- (from NADH) = H+ + 2 e-

(bound to 1st carrier) then e- flow through at least 15 different electron carriers; in the process,

energy released by e-used to move H+.

-use step wise process because if just let e- go from NADH to O2 directly, would be so favorable

it would cause an explosion

- 3 Major Complexes:

a) NADH dehydrogenase complex (at least 12 polypeptides) transfers e- to ubiquinone

b) b-c1 complex (8 polypeptides), probably present as dimer - receives e- from ubiquinone and

transfers them to cytochrome c

c) Cytochrome Oxidase Complex (7 polypeptides), dimer - uses 4 e- from cytochrome c and O2

to produce 2xH2O

- 3 major complexes, Ubiquinone (also called coenzyme Q) and cytochrome c diffuse in plane

of membrane as individual entities. Vectorial organization

-organized in order, but not stuck together, kind of flowing laterally and bump into each other

- On the average, for each NADH oxidized, 2e- transferred, 3ATP (maximum) synthesized.

E- Flow:

NADHNADH dehydrogenaseubiquinonecyt. b-c1 complexcyt. ccyt. oxi. complexO2

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