Cellular Organization

Peter TakizawaDepartment of Cell Biology

•Organelles

•Distribution of organelles by microtubules and motor proteins

•Signal sequences

• Importing proteins into organelles

Cells contain a variety of organelles that perform specific and unique functions.

Organelles are collections of specific proteins and other components. Each type of organelle performs a unique set of biochemical reactions. Organelles increase the efficiency of these reactions by concentrating the proteins and substrates of a common pathway in small environment. Some organelles are surrounded by a membrane similar to the cell membrane which helps create a specific internal environment. For example, the lysosome has an acidic pH compared to the cytoplasm.

Lysosomes contain a variety of digestive enzymes that would chew up cell components.

NucleasesProtease

GlycosidasesLipases

Lysosome

Another advantage of organelles is that they protect cells from noxious molecules. The lysosome contains many digestive enzymes that breakdown old or foreign macromolecules, including protein, nucleic acids and carbohydrates. If these enzymes were released to the cytoplasm, they could digest critical cellular components. By housing these enzymes in a membrane-bound organelle, the cell not only increases the efficiency of the digestive reactions but also protects the rest of the cell.

Organization of the cytoplasm: microtubules and actin filaments

Cells use microtubules to position organelles within their cytoplasm.

Cells organize and localize organelles within their cytoplasm by moving them to intracellular locations where they are needed. For example, cells will position mitochondria at sites of greatest energy consumption. To position organelles, cells need a mechanism to transport organelles through the cytoplasm and then anchor them at specific sites. Microtubules play a critical role in organizing cytoplasm of cells as shown in these images. Microtubules are in red and Golgi, a membrane-bound organelle, is in green. Cells utilize microtubules to tether Golgi in specific location in the cell, usually adjacent to the nucleus. If we disrupt the microtubules with a drug, the Golgi fragment and lose their positioning.

Microtubules extend throughout the cytoplasm.

Microtubules are effective for organizing cells because they are long filaments that extend throughout cytoplasm. They are large enough to span most cells and allow cells to position organelles in specific regions. Microtubules are labeled in green.

Microtubules are a hollow tube of tubulin dimers.

_-tubulin

`-tubulin +

-Microtubules are composed of a heterodimer of alpha and beta tubulin. Both bind the nucleotide GTP. Heterodimers assemble into filaments end on end to form protofilaments. 13 protofilaments interact to make a single microtubule. The arrangement of the protofilament creates a long, hollow tube similar to a PVC pipe. The extensive lateral contacts between subunits in neighboring protofilaments gives microtubules greater strength and allows them to grow longer than actin filaments. Microtubules are also polarized with one end, the minus end, containing an exposed alpha subunit and the other end, the plus end, having an exposed beta subunit. Microtubules grow in length from their plus ends, but their minus ends are unstable and are usually associated with proteins that prevents subunits from depolymerizing from the filament.

Microtubules grow from their plus ends.

Video shows growth of microtubules in vitro. Microtubules grow from their plus ends as more dimers are added to the filament. Occasionally, microtubule will stop growing and then shrink as dimers fall off of plus end. Inside cells, the plus ends of microtubules are capped or stabilized so they don’t shrink. The repeated growing and shrinking allow microtubules to explore different areas of the cell and cells can reposition or reorient microtubules. Note that the minus ends of the microtubules emanate from a common center. This region is called the microtubule organizing center and stabilizes the minus ends of microtubules.

Proteins link organelles to microtubule network.

The mitotic organizing center (MTOC) stabilizes the minus ends of microtubules in most cells. The MTOC contains a large number of proteins including a pair of organelles called centrioles. Centrioles are an example of an organelle that is not membrane-bound. The location of the MTOC determines the orientation of microtubules in a cell. If the MTOC is located in the center of the cell, the microtubules radiate outward toward the cell membrane with their plus ends at the cell membrane.

Kinesins and dynein mediate bidirectional transport of organelles along microtubules.

Dynein

Microtubules also mediate the transport of organelles, proteins and nucleic acids within the cytoplasm of cells. Microtubules-based motor proteins, kinesins and dynein, use microtubules to move cellular material within cells. Kinesins comprise a large family of motor proteins most of which move toward the plus ends of microtubules. In contrast, dynein moves toward the minus ends. All motor proteins contain a domain that binds microtubules and hydrolyzes ATP. The energy released through ATP hydrolysis propels the motor protein along a microtubule. Motor proteins that transport cellular material also contain a domain that interacts specifically with an organelle, protein or nucleic acid. By controlling the type of motor protein is attached to an organelle, cells control the location of that organelle.

Cells regulate activity of motor proteins to control distribution of organelles.

Organelles will often have both types of motors on their surface, allowing cells to adjust their position. For example, these melanocytes contain a pigmented organelle called a melanosome. In some organisms, melanocytes position melanosomes in response to the amount of light. In the presence of light. kinesin on the surface of melanosomes moves them to the periphery of cells. In the dark, dynein returns the melanosomes to the center of the cell.

Actin filaments concentrate at the plasma membrane in many cells.

Microtubules

Actin Filaments

Nucleus

Actin filaments show a different distribution than microtubules. Actin filaments usually localize near the cell membrane or at sites where cells attach to another cell or external surface. Actin filaments provide structural support to the cell membrane and allow cells to generate tension on the cell membrane which can lead to cell contraction.

Actin filaments are a polymer of a single protein.

Actin filaments are a polymer of a single globular protein of about 43 kD. Actin monomers bind and hydrolyze ATP and have an asymmetric structure as one side of the protein contains the pocket to bind ATP. Actin monomers polymerize into filaments in a helical fashion. Actin filaments appear to be of two single filaments wrapped around each other. The lateral interactions between monomers allows filaments to grow to greater lengths than if the filament were a single, straight chain of monomers. Individual actin filaments are shorter and less rigid than microtubules.

Some types of myosins transport vesicles and organelles.

Similar to microtubules, actin filaments also support the transport of organelles, proteins and nucleic acids. Instead of kinesins and dynein, actin filaments use myosins to transport intracellular organelles and other cellular material. These myosins resemble the structure of kinesin in that contain motor domains that bind actin filaments and hydrolyze ATP and a domain at their C-terminus that links the motor to different cargo. The motor domains of myosin generate force along actin filaments to move themselves within the cytoplasm.

Microtubules support long distance transport; actin filaments transport near the cell membrane.

Kinesin

Myosin

Because actin filaments are shorter than microtubules, myosins usually transport organelles over shorter distances compared to kinesins and dynein. Myosin-based transport is often used near the plasma membrane, an area rich in actin filaments. One model of how kinesins and myosins work together is that kinesins transport organelles from the center of the cell towards the periphery, where myosins take over moving organelles near the plasma membrane. For example, a secretory vesicle might be transported from the trans-Golgi network towards the plasma membrane along microtubules. When the secretory vesicle reached the actin filaments underneath the plasma membrane, myosins would complete the transport of the vesicle to the plasma membrane.

Targeting proteins to specific organelles

Proteins synthesized in cytoplasm must get to correct organelle.

Each organelle contains a unique set of proteins and other components that allows that organelle to perform its biochemical functions. Cells need a mechanism to target certain proteins to one type of organelle. In addition, because many organelles are surrounded by a membrane, there must be a way for proteins to cross the membrane of an organelle.

Proteins are imported into organelles from cytosol or transported between organelles.

Cytosol

Golgi

Endoplasmic Reticulum

Late Endosome

Lysosome

Plasma Membrane

Secretory Vesicles

PeroxisomesMitochondriaNucleus

Signal sequences target proteins to specific organelles. Signal sequences are usually one or more stretches of amino acids within a protein that is recognized by machinery that delivers that protein to an organelle. The synthesis of all proteins starts in the cytosol, but those proteins with signal sequences are delivered to their appropriate organelle. Many organelles receive proteins through the secretory pathway. Proteins are initially translated and inserted into the ER. Proteins are transported to the Golgi and then delivered to the different organelles. Some organelles receive proteins directly from the cytosol. The nucleus is a unique organelle as it allows bidirectional movement of proteins.

Proteins contain signal sequences that determine their final destination.

Function of Signal Sequence Example of Signal Sequence

Import into nucleus -Pro-Pro-Lys-Lys-Lys-Lys-Lys-Val-

Export from nucleus -Leu-Ala-Leu-Lys-Leu-Ala-Gly-Leu-Asp-Ile

Import into mitochondria+H3N-Met-Leu-Ser-Leu-Arg-Gln-Ser-Ile-Arg-Phe-Phe-Lys-Pro-Glu-Arg-Asn-Thr-Leu-Cys-Ser-Ser-Arg-Tyr-Leu-Leu

Import into ER+H3N-Met-Met-Ser-Phe-Val-Ser-Leu-Leu-Leu-Val-Gly-Ile-Leu-Phe-Trp-Ala-Thr-Glu-Ala-Glu-Gln-Leu-Thr-Lys-Cys-Glu-Val-Phe-Gln

Import into peroxisomes -Ser-Lys-Leu-COO-

Signal sequences that target proteins to the different organelles have been identified. Some signal sequences use a nearly identical stretch of amino acids in different proteins to target those proteins to a specific organelles. Other signal sequences use the properties of amino acids (hydrophobic, acidic, basic) to target proteins to an organelle. The amino acids in these signal sequences will often vary between proteins.

Small changes in signal sequences have profound effects on location of proteins.

HumanNucleosideTransporter

PEXN

MouseNucleosideTransporter

PARS

Mitochondria

PlasmaMembrane

Slide illustrates how subtle changes in signal sequences can affect the localization of proteins. In humans, a certain nucleoside transporter contains a 4 amino acid motif that targets it to mitochondria. In mice, the same protein contains two changes in this motif and this targets the protein to the plasma membrane. If you experimentally change the motif in the human protein to the mouse version, the human transporter localizes to the plasma membrane.

Fialuridine is a uridine analog that is a potent inhibitor of hepatitis B.

Human Mitochondrion

Mouse Mitochondrion

NucleosideTransporter

In the early 1990’s there was a clinical trial of a drug designed to treat hepatitis B in 15 patients. Hepatitis B is a virus that affects the liver, causing inflammation. One common treatment for this type of virus is nucleoside analogs. Nucleoside analogs are similar to the nucleosides that are used to synthesize DNA, so they are often incorporated into DNA, but the analogs are altered so that they stop synthesis or affect the stability of the DNA. Fialuridine is a nucleoside analog that was found to be effective in reducing hepatitis B in animal models. It was subsequently used in human trials and initially it appeared to reduce the amount of virus. But then something went terribly wrong. Long after the clinical trials were concluded, researchers discovered something unique about the way fialuridine affects human cells. Because humans have a certain nucleoside transporter that localizes to mitochondria, fialuridine can enter mitochondria. Why is this bad? Mitochondria contain their own DNA genome and require expression of genes in their genome to function properly. By preventing replication of the mitochondria genome, fialuridine poisoned mitochondria, reducing their ability to generate ATP. Why wasn’t this discovered before the clinical trials? Fialuridine was tested on animal models for safety, but those animals were mice and rats whose nucleoside transporter lacks the mitochondrial signal sequence. Consequently, their mitochondria did not take up fialuridine and were not affected by the drug.

What happened in the clinical trials? By reducing the activity of mitochondria, fialuridine forced energy-intensive cells to start using glycolysis to generate ATP. This lead to an increase in lactate in the blood and a condition called lactic acidosis. Because the liver processes lactate, the increase production of lactate led to liver failure 7 patients, 5 of whom died.

Importing proteins into membrane-bound organelles

So we’ve seen how proteins get targeted to a membrane-bound organelle but how are they imported into that organelle. Proteins can’t diffuse across the membrane so they need a way to get across. Organelles that directly import proteins from the cytoplasm have proteins that form pores in their membrane. These pores allow proteins to transit from the cytoplasm into the organelle. We’re going to look at two different types of pores.

Most proteins imported co-translationally into the ER through the translocator.

Signal sequence

The ER contains a set of proteins called the translocon that form a pore in the membrane through which proteins will be threaded from the cytoplasm into the ER. Two important notes about the process of moving a protein from the cytoplasm to ER. First, the translocation of proteins is usually coupled to their synthesis. As mRNAs are being translated the newly made protein is being inserted into the translocon. The energy used during translation can push the protein through the translocon. The second important point is that proteins cross the ER in an unfolded state. The translocon is not large enough to accommodate folded proteins. Consequently, proteins must fold into their 3 dimensional structure in the ER. As we will see in future lectures, the need for proteins to fold in the lumen has some important medical consequences. In this cartoon the signal sequence of the inserted protein is cleaved, generating a protein that resides in the lumen. But we also need a way to make integral membrane proteins.

Integral membrane proteins contain stop transfer sequence.

N-terminus

C-terminus

Cytosol

Lumen

Signal sequence

Stop transfer sequence

Integral membrane proteins contain a stretch of hydrophobic residues downstream from the signal sequence that function as stop transfer sequence. The stop transfer sequence halts the translocation of the protein. Eventually the protein will exit from the translocon and the stop transfer sequence will function as the transmembrane domain of the protein.

Nuclear pores are size selective channels.

The nucleus also uses a pore to import proteins, but uses a different mechanism than the ER. First, nuclear pores are large and can accommodate folded proteins. In fact, they can accommodate large multi-protein complexes such as ribosomes. The pores are so large than small proteins (< 30 kDa) can freely diffuse through the pore.

Importins bind import signal sequences and mediate passage through nuclear pores.

Importin

Signal sequence

How do proteins larger than 30 kD get into the nucleus. These proteins have a signal sequence called the nuclear localization sequence. The NLS is recognized in the cytoplasm by a protein called importin. Importin binds the NLS and then guides the proteins to the nuclear pore. The complex then migrates through the pore to enter the nucleus. Inside the nucleus the complex is dissociated allowing the imported proteins to perform its function in the nucleus.

Protein import into peroxisomes and Zellweger’s Syndrome

Peroxisomes detoxify chemicals, metabolize fatty acids and synthesize key lipid.

Peroxisomes perform several vital functions. They metabolize many harmful molecules, such as phenols, formaldehyde and ethanol. They metabolize fatty acids into acetyl CoA which can be used in metabolic pathways to generate ATP. Finally, peroxisomes catalyze the initial step in the synthesis of a special lipid called plasmalogen. Plasmalogen localizes to the cell membrane of cells that form myelin sheaths around axons.

Proteins inserted post-translationally into peroxisomes.

© 2002 Nature Publishing Group384 | MAY 2002 | VOLUME 3 www.nature.com/reviews/molcellbio

P E R S P E C T I V E S

nsPME–PEX5 complexes shortly after theirsynthesis but before their import. We referto these pre-import complexes of nsPMEsas ‘preimplexes’.

We see preimplex formation as a stochasticprocess that is controlled primarily by the con-centrations of the relevant proteins in thecytoplasm and their affinities for one another(FIG. 2). In such a system, there are many waysfor nsPMEs to interact with preimplexes. Forexample, peroxisomal enzymes that oligomer-ize rapidly and/or have a low affinity for PEX5are more likely to oligomerize before theyenter preimplexes, whereas proteins thatoligomerize more slowly and/or have a rela-tively high affinity for PEX5 might enter thepreimplex as monomers. Even monomericproteins should enter preimplexes, providedthey contain a PTS1. An intrinsic part of thepreimplex hypothesis is that some nsPMEsmust oligomerize before, or during, preim-plex formation, otherwise the nsPME–PEX5interactions would not be mutually multiva-lent. However, various factors (enzyme con-centration, degree of enzyme oligomerizationand affinity of the PTS1 of each enzyme forPEX5) make it difficult to predict exactly whatproportion of peroxisomal enzymes has tooligomerize before, or during, preimplex for-mation in order for it to proceed.

Another prediction of the preimplexhypothesis is that other PEX5-binding pro-teins might have a significant effect on preim-plex dynamics. For example, PEX14 is partic-ularly suited to having such a role. PEX14 isthe primary PEX5-docking site in the peroxi-some membrane, and it is a dimeric proteinwith at least one high-affinity PEX5-bindingsite per monomer29,34. Each PEX5 monomerhas several PEX14-binding sites, and soPEX5–PEX14 interactions are also mutuallymultivalent. In addition to helping tetherpreimplexes to the peroxisome surface,PEX5–PEX14 interactions might actually pro-mote preimplex expansion by linking other-wise distinct nsPME–PEX5 complexes atPEX14 dimers (FIG. 2).

Although the biochemical properties ofperoxisomal enzymes, PEX5 and PEX14 indi-cate that preimplex assembly is probable, whatpossible benefit is derived from assemblinglarge nsPME–PEX5 complexes on the peroxi-some surface before nsPME translocation?One possibility is that the multivalent natureof nsPME–PEX5–PEX14 interactions allowsthe rapid and specific delivery of nsPMEs tothe peroxisome using only diffusion and thebinding energy of these protein–protein inter-actions. Such a mechanism might explain whyPEX5-docking factors are the only proteinsthat have been implicated in delivering PEX5

and X-ray-crystallography structure datahave shown that PEX5 binds one PTS1 perPEX5 monomer, and that PEX5 behaves asan oligomer — most probably a tetramer —with several PTS1-binding sites10,29. On itsown, the fact that PEX5 is multivalent withrespect to its PTS1 ligands is not remark-able. However, the fact that PEX5 can bindseveral nsPMEs becomes more intriguingwhen we consider that peroxisomes canimport oligomeric peroxisomal enzymes2–4,and most, if not all, peroxisomal enzymesare oligomers30–33. So, the high-affinity inter-actions between PEX5 and nsPMEs (Kd =~100 nM)10 are likely to be mutually multi-valent. On the basis of these considerations,we propose that nsPMEs form large

nsPME import, but less is known about theirpoint of action11.

This general model of peroxisomal-matrix-enzyme import has been useful forassigning general functions to several of theknown peroxins, but suffers from a lack ofmechanistic detail. For example, peroxisomal-matrix-enzyme import requires ATP27,28, andthis model makes no predictions about whereATP is consumed or how ATP hydrolysis pro-motes enzyme translocation. This model alsofails to explain how peroxisomes importfolded, oligomeric enzymes across a sealedmembrane2–4. In light of these limitations, wehave re-examined the existing data on peroxi-somal-matrix-protein import from a mecha-nistic perspective. The remainder of this arti-cle presents a more detailed description ofwhat has arisen from our reconsideration ofthese data.

The ‘preimplex’ hypothesisTo gain insight into the molecular mecha-nisms of matrix-protein import, we concen-trated our attention on those aspects of per-oxisomal-matrix-protein import for whichthe biochemical data are most reliable. Webegan with the recognition of PTS1-con-taining enzymes by PEX5, the step inimport that has been subjected to the mostrigorous biochemical analysis. Biochemical

Figure 1 | A general model of peroxisomal-matrix-enzyme import. The dynamic distribution of PEX5indicates that matrix-enzyme import involves: a | binding of enzymes (red circles) by the import receptors(shown here as PEX5 in green); b | transport of receptor–enzyme complexes to the peroxisome surface; c | docking of receptor–enzyme complexes through protein–protein interactions with peroxisomalmembrane proteins, such as PEX14 (purple) and, perhaps, PEX13 (grey); d | dissociation ofreceptor–enzyme complexes and enzyme translocation through a proteinaceous pore (perhaps containingPEX8, PEX10 and PEX12, all shown in blue); and e | receptor recycling, which might involve PEX4 andPEX22 (both shown in orange). PEX, peroxin.

PEX5

PEX14 PEX13PEX8

PEX12 PEX10

PEX4

PEX22

a Binding

b Transport

c Docking

d Translocation

e Receptor recycling

Cytosol

Peroxisome

“We see preimplexformation as a stochasticprocess that is controlledprimarily by theconcentrations of therelevant proteins in thecytoplasm and their affinitiesfor one another.”

Protein import into peroxisomes is similar to other membrane-bound organelles in that peroxisomal proteins contain a signal sequence that is recognized by proteins that deliver the peroxisomal protein to the peroxisome. Another set of proteins in the peroxisome membrane forms a channel through with peroxisomal proteins enter a peroxisome. The proteins that target proteins to peroxisomes and form the channel are called Pex proteins.

Mutations in Pex proteins lead to loss of peroxisomes.

41

Fig. 1A–H PEX1 expressionrestores peroxisome biogenesisin CG1 patient cell lines. Cul-tured cells were transfectedwith either control pcDNA3plasmid (left panels: A, C, E,G) or the pcDNA3-PEX1 plas-mid pBER81 (right panels: B,D, F, H) and analysed by indi-rect immunofluorescence toperoxisomal matrix proteins byusing an anti-SKL antibodyand a Texas-Red-labelled goatanti-rabbit secondary antibody.A, B Patient 4065, C, D pa-tient 2775, E, F patient 3488,G, H patient 1742. PEX1 celltransfection efficiency was11%–18%

Fig.2A, B PEX1 exon 13base insertion in CG1 patients.A Automated sequence chro-matogram of a region of exon13 amplified by PCR from ge-nomic DNA of patient 4756, aheterozygote for the exon 13insT mutation. Left Subclonedwild-type allele, right sub-cloned exon 13 insT allele.B PEX1 protein sequence en-coded by the wild-type (WT)and exon 13 insT (Ex13insT)alleles. Histidine 690 is num-bered. Tyrosine 700 of the mu-tant sequence is underlined toindicate the position of theframeshift from the exon 13insT allele. Asterisk Termina-tion codon

pex1 Normal

Cells that contain mutations in the Pex genes can’t import porteins into peroxisomes. Consequently, the protein content of peroxisomes can’t be maintained and the cell loses its peroxisomes.

Pex mutations cause Zelleweger Syndrome and demyelination of nerves.

There are several diseases, termed Zelleweger Syndrome, that are caused by mutations in the genes that encode the Pex proteins. These mutations lead to a loss of peroxisomes. The consequence is a build up of toxic material in cells, especially in the liver, and an inability to fully myelinate the axons of neurons. The lack of myelinated axons affects motor neurons and infants with Zelleweger Syndrome have low muscle tone, show an inability to move and often fail to suckle. The prognosis of infants with Zelleweger Syndrome is poor and most die by 6 months.

Take home points...

• Organelles increase efficiency of biochemical reactions and protect cells from noxious molecules.

• Cells control the distribution of organelles through motor proteins and the cytoskeleton.

• Signal sequences guide proteins to specific organelles where protein machinery imports them into the organelle.