17 March...

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Vesicle Transport

Vesicle Formation • Curvature (Self Assembly of Coat complex) • Sorting (Sorting Complex formation) • Regulation (Sar1/Arf1 GTPases) • Fission ()

Membrane Fusion • SNARE combinations • Tethers • Regulation by Rabs • SM Proteins

Vesicle pathway: many compartments, interconnected by trafficking routes

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Two Key Steps: ���1. sorting during vesicle formation���2. targeting during vesicle fusion

Morphological Experiments Suggest Role of Vesicle Coats

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Each type acts at distinct locations

Components that participate in budding of coated vesicles"

Figure 17-51!

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Clathrin coated vesicles

Clathrin cycle

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Dynamin pinches of the vesicles

GTPase

Shibire mutant has coated pits but no budding off of synaptic vesicles

GTP hydrolysis by dynamin is required for pinching off of clathrin-coated vesicles "

Figure 17-55!

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Clathrin adaptins and dynamin

Figure 7.22

Solutes

Pseudopodium

“Food” or other particle

Food vacuole

CYTOPLASM

Plasma membrane

Vesicle

Receptor Ligand

Coat proteins

Coated pit

Coated vesicle

EXTRACELLULAR FLUID

Phagocytosis Pinocytosis Receptor-Mediated Endocytosis

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Figure 7.22a

Pseudopodium

Solutes

“Food” or other particle

Food vacuole

CYTOPLASM

EXTRACELLULAR FLUID

Pseudopodium of amoeba

Bacterium Food vacuole

An amoeba engulfing a bacterium via phagocytosis (TEM).

Phagocytosis

1 µ

m

Figure 7.22b

Pinocytosis vesicles forming in a cell lining a small blood vessel (TEM).

Plasma membrane

Vesicle

0.5 µ

m

Pinocytosis

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Figure 7.22c

Top: A coated pit. Bottom: A coated vesicle forming during receptor-mediated endocytosis (TEMs).

Receptor

0.25

µm

Receptor-Mediated Endocytosis

Ligand

Coat proteins

Coated pit

Coated vesicle

Coat proteins

Plasma membrane

The macromolecules that are degraded in the lysosome arrive by endocytosis, phagocytosis, or autophagy.

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When the protein is properly folded, COPII coated vesicles transport the proteins via the vesicular tubular cluster (vtc) to the cis-Golgi network.

• The COPII coating is removed (Sar1 hydolyzes GTP) and the vesicles fuse with each other to form the vtc. • The vtc is motored along microtubules that function like railroad tracks. • The vtc fuses with the cis-Golgi network.

Transport from the ER to the Golgi

Some proteins exiting the ER are returned to the ER by COPI coated vesicles. These proteins are identified by the presence of specific signal sequences that interact with the COPI vesicles or associate with specific receptors.

Example of retrieved protein: ER chaperones like BiP that are mistakenly transported.

This example describes the situation of BiP. BiP has the signal sequence, KDEL. When BiP escapes the ER, it associates with the KDEL receptor. The slightly acid environment of the vtc and Golgi favor this association. When the returning vesicle fuses with the ER, the neutral pH of the ER causes BiP to dissociate from the receptor.

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Proteins exiting the ER join the Golgi apparatus at the cis Golgi network. The Golgi apparatus consists of a collection of stacked compartments.

nucleus

Cell membrane

The Golgi Apparatus has two major functions: 1. Modifies the N-linked oligosaccharides and adds O-linked oligosaccharides. 2. Sorts proteins so that when they exit the trans Golgi network, they are delivered to the correct destination.

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Modification of the N-linked oligosaccharides is done by enzymes in the lumen of various Golgi compartments.

While N-linked glycosylation appears to function in protein-folding in the lumen of the ER, the function of the oligosaccharide modifications occurring in the Golgi is largely unknown.

One ultimate destination of some proteins that arrive in the TGN is the lysosome. These proteins include acid hydrolases.

Lysosomes are like the stomach of the cell. They are organelles surrounded by a single membrane and filled with enzymes called acid hydrolases that digest (degrade) a variety of macromolecules. A vacular H+ ATPase pumps protons into the lysosome causing the pH to be ~5.

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The macromolecules that are degraded in the lysosome arrive by endocytosis, phagocytosis, or autophagy.

The acid hydrolases in the lysosome are sorted in the TGN based on the chemical marker mannose 6-phosphate.

This was first attached in the ER.

The phosphate is added in the Golgi

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Adaptins bridge the M6P receptor to clathrin.

Hydrolases are transported to the late endosome which later matures into a lysosome.

Acidic pH causes hydrolase to dissociate from the receptor.

Guidance of vesicular transport SNAREs: specificity and fusion Rab GTPases: initial docking and tethering of vesicles to target membranes and matching of v- and t- SNAREs

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SNARE proteins guide vesicular transport 20 SNAREs, vesicle-SNAREs, target-SNAREs

SNAREs specify compartment identity and control specificity

4 α helices in trans-SNARE complexes

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Rab proteins ensure the specificity of vesicle docking

>30 Rabs On cytosolic surface

C-terminal regions are variable: Bind to other proteins, including GEFs

SNAREs may mediate membrane fusion

SNARE complex

After docking

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The entry of enveloped viruses into cells

HIV

Similar to SNAREs

Proteins leave the ER in COPII-coated transport vesicles

ER exit sites (no ribosomes)

Selective process

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Only properly folded and assembled proteins can leave the ER

Chaperones cover up exit signals

Homotypic membrane fusion

to form vesicular tubular clusters

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Vesicular tubular clusters

Lacks many of the ER proteins

COPI-coated

Retrograde transport:

Short-lived

carry back the ER resident proteins that “leaked” out

ER retrieval signals: KKXX in ER membrane proteins, KDEL sequence in soluble ER resident proteins

Membrane proteins in Golgi and ER have shorter TM domains (15 aa) Cholesterol

pH controls affinity of KDEL receptors

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Ordered series of Golgi compartments

Cisternae, tubular connections

Plant cell

Two main classes of N-linked glycosilation

core complex oligosaccharides

high-mannose oligosaccharides

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Oligosaccharide processing in the ER and the Golgi

Histochemical stains: biochemical Compartmentalization of the Golgi

Functional compartmentalization

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Transport through the Golgi may occur by vesicular transport or cisternal maturation (not mutually exclusive)

Collagen rods- SUBJECT OF PAPER Scales in algae

Two Key Steps: sorting during vesicle formation & targeting during

vesicle fusion

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Summary 1.  Vesicular transport, biosynthetic-secretory and

endocytic pathways; 2. Coated vesicles; 3.  Coat assembly and disassembly, budding, dynamin,

coat-recruitment GTPases; 4.  Targeting and fusion by Rab GTPases, SNAREs; 5.  ER to Golgi: COPII, folding, fusion (cluster), retrograde; 6.  Golgi apparatus structure and polarity; 7.  Continuation of glycosylation; 8.  Compartmentalization of Golgi cisternae.