Chapter 15. Overview of Vesicular Transportlewisj/Cell.04/Lectures/Chap15b.6.pdfOverview of Vesicular Transport •Vesicle formation, targeting and fusion are separate events, each

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Chapter 15. Overview of Vesicular Transport

• Proteins do not normally make multipletransits through a membrane.

• Therefore, once made in the ER they movefrom compartment to compartment viavesicular transport.

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• These steps retain membrane polarity.• They also retain the separation of cytosolic

and non-cytosolic compartments.• These steps transfer both contents and

membrane. Review.

Fig 11.19

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• Steps in vesicular traffic. (Fig 15-17)

Fig 15.174


• Vesicle formation, targeting and fusion areseparate events, each with their ownbiochemistry.

• Bulk flow vs. sorting - an importantdifference in cargo transport via vesiculartransport.

• An analogy -- the widget factory.

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• There are different types of coated vesicle.– Clathrin-coated– Non-clathrin coated

• COP-coated

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Chapter 15. Overview of Vesicular Transport.• Vesicular traffic involves vesicles with a

cytosolic protein layer (coated vesicles).Cargo is composed of membrane componentsand/or content of the vesicle.

non-cytosolic

cytosolicmembrane coat

cargo

Fig 15.18

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Chapter 15. Mechanism of Vesicle Formation

• The best understood example of vesicleformation is the clathrin coated vesicle.

• Involved in sorting particular cargo awayfrom other cargo.

• The model - Figure 15-19.

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Fig 15.19

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• Clathrin will self-assemble in vitro.• Self-assembly results in curved or spherical

cages.• Thus, clathrin supplies the brawn.

• Movie

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• In vivo, clathrin assembly is promoted by“assembly proteins”– Monomeric protein (AP 180)

• Co-assembles with the clathrin.• Strongly promotes assembly.

– Adaptin complex (or related molecules)• Localizes to particular membrane• Binds to receptors.• Promotes assembly• (The “brains” of clathrin formation). 12


• Adaptins and related molecules:– Adaptin AP2 (plasma membrane, endocytosis)– Adaptin AP-1B (TGN to plasma membrane)– Adaptin AP-1A (Endosome to TGN)– GGAs (TGN to endosome)

• GGA is related to part of the AP-1 complex, althoughit is not itself part of an AP complex

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Boehm &Bonifacino.2001, Mol.Biol Cell12:2907-20.

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Chapter 15. Mechanism of VesicleFormation

• Formation of COP-coated vesicles.– Coatomer proteins are involved on the cytosolic

side of the membrane.– Does not involve clathrin or adaptors but does

make use of dynamin.– Unlike clathrin, coat remains until vesicles reach

target.– COP I is probably associated with vesicles

formed in the ER and COP II with the Golgiformed vesicles.

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Chapter 15. Mechanism of Vesicle Fusion

• SNARES are involved in targeting

Fig. 15.2016

Chapter 15. Mechanism of VesicleTargeting

• Snares are presumed to be involved in allvesicle transport steps, including COP-coatedvesicles not just the clathrin coated onesshown here.

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• The thermodynamic barrier to fusion.

• Where have we seen logically similarproblems before?

• What was the solution?

• The importance of fusigns18


• SNARES appearto drivemembrane fusionas well asfunction indocking.

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• SNARES appear to drive membrane fusionas well as function in docking.

Big Alberts, 4th ed.Figs 13-12, 13-13

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• The importance of viral fusigens forscience.

– Why it makes sense to look for viral fusigens.

– Several fusigens have been clearly identified,and the mode of action has been largely workedout.

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Chapter 15. Flow from ER to Golgi

• Material flows automatically from ER toGolgi if it passes ER quality control. This isan example of bulk flow.

• This involves non-clathrin coated vesicles.

• ER resident proteins go along with this bulkflow but are brought back from Golgi inanother set of non-clathrin coated vesicles. 22


• Most evidence for flow from ER to Golgi isbiochemical.

• However the following experiment showsdirect transport. John F. Presley. Nelson B. Cole,Trina A. Schroer, Koret Hirschberg, Kristien Zaal andJennifer Lippincott-Schwartz. 1997. ER to Golgi trafficvisualized in living cells. Nature 389: 81-85.

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• The probe: the gene for GFP was attachedto the gene for the C-terminus of VSVG, atemperature sensitive viral glycoproteinwhich moves through the secretory pathwayto the cell surface. GFP is a “glow in thedark” protein that comes from certainjellyfish. Thus the glow tells you where theprotein is located.

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• The experiment: Cells were transfectedwith the probe and allowed to synthesize thechaemeric protein under temperatures thatprevented the protein from entering theGolgi

• The temperature was then shifted to allowsuch transport.

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• A modification:

• Here the protein associated with the Golgiwas photobleached and new protein getsmoved into the cis Golgi.

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Chapter 15. The Golgi Apparatus

• The Golgi apparatus is a modifying andsorting machine.

• Similar to a factory line.– Material is loaded in at one end (the cis or

forming face)– Material is removed in the other (the trans or

maturing face)– Material is sequentially processed in between.

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• The Morphology of the Golgi.– Closed set of sacks.– Stacked sacks.– Receives material via vesicles at one side and

ships out material via vesicles (usually) at theother.

– Specific enzymes are located in specificcisternae.

– See figure 15-2430Fig. 15.24

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• Examples of processing material flowingthrough the Golgi.– O-linked glycosylation.– Protein cleavages.– Processing of N-linked oligosaccharides

originally added in the ER.

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Supplement: Which way to the Golgi?

• See Featherstone, C. 1998. Coming to gripswith the Golgi. Science 282:2172-2174 formore information.

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Supplement: Which way the Golgi?

• I. There is no doubt that material flowsfrom the ER --> cis Golgi --> medial Golgi -->trans Golgi --> and out.

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• II. Two models for this process.– A. Vesicular transport

• 1. Vesicles bud from ER, fuse with cis cisterna.• 2. Vesicles bud from cis cisterna, fuse with medial

cisterna.• 3. Vesicles bud from medial cisterna, fuse with trans

cisterna.• 4. Key concept: the cisterna itself is a

permanent structure through which material (bothmembrane and lumenal contents) flows.

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• II. Two models for this process.– A. Vesicular transport

• 5. Analogy: a factory line with stationary workers ina stationary building. Material is carried tosuccessive buildings by a conveyer belt. In eachbuilding a different team of workers carries out itsjob in processing the product before the materialmoves to a different building, with a different team.

• 6. This is the way a pickle factory or automobile lineworks.

• 7. The model used by your text. 36


• II. Two models for this process.– B. Cisternal maturation model

• 1. Vesicles bud from ER, fuse with cis cisterna.• 2. Cis cisterna matures into a medial cisterna, new

cis cisterna forms behind it.• 3. Medial cisterna matures into a trans cisterna, new

medial cisterna matures behind it.• 4. Key concept: cisternae (with both membrane and

contents) start out as cis cisterna and develop intotrans cisterna)

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Supplement: Which way the Golgi?• II. Two models for this process.

– B. Cisternal maturation model• 5. Analogy: a factory line where entire room, product

and workers move along the conveyer belt. Atintervals, a team of workers boards a bus and moves toan more recently constructed building with lessdeveloped product. They might carry some of thebuilding materials with them for use in the newbuilding.

• 6. This is the way Boeing makes airplanes.

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• III. Why can’t people observe the processdirectly?– A. Most biochemical methods show where

material is at a given time, but not how it gotthere.

– B. The Golgi and vesicles are too small to beeasily observed by light microscopy.

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• III. Why can’t people observe the processdirectly?– C. Preparing material for electron microscopy

kills the cells and therefore must stop thisprocess. Therefore, electron microscopy cannoteasily resolve this question.

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• IV. Information that relates to the models– A. Although material moves through stacks,

many enzymes (and thus many enzyme mediatedreactions) are found only in the cis cisterna,others only in medial cisterna and some othersonly in trans cisterna.

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• IV. Information that relates to the models– A. Reactions are localized in particular

cisternae.• 1. In the vesicular transport model, the enzymes just

have to stay home while the material moves fromcisterna to cisterna.

• 2. In the cisternal maturation model, enzymes thatfunction in the cis cisterna must continually be movedas one cisterna matures into a medial cisterna andanother cis cisterna forms. A similar process musthappen for enzymes/reactions throughout the stack. 42


• IV. Information that relates to the models– B. Material can move between stacks by

vesicular transport.• 1. This is a requisite for the vesicular transport

model where they would be involved in forwardtransport of materials.

• 2. Could also be explained by reverse transportunder the cisternal maturation model. Suchtransport must be responsible for maintaining thedistribution of enzymes and other molecules in theparticular kind of cisterna in which they reside (seeabove).

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• IV. Information that relates to the models– C. Abundant vesicles are located near the

sides of a Golgi stack.• 1. In the vesicular transport model they could

be involved in forward transporting material betweenthe cisterna.

• 2. In the cisternal maturation model they couldbe involved in the reverse transport moving enzymesand other molecules, as described above.

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• IV. Information that relates to the models– D. Scale formation in algae. In some algae

scales are built in the Golgi and then secretedout to make a cell covering. The scales areclearly built up in this process and are too largeto fit in a transport vesicle.• 1. This is a impossible one for the vesicular

transport model to explain, although many haveassumed this is a particular and unusual case.

• 2. The cisternal maturation is supported by thisobservation at least in this case.

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• V. Final notes:– The cisternal maturation model used to be widely

accepted (in fact an earlier word for the “cis”face (=side) of the Golgi was the “forming face”and an earlier word for the “trans” face (=side)of the Golgi was the “maturing face”. Then thevesicular transport model took over (with namechanges in the Golgi cisternae) and has been theconventional wisdom for many years now.Recently the question is being reexamined.

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• V. Final notes:– Note how “facts” and “information” can look

very different depending on which one modelone places it in. For example, the experiment inPanel 13-1a was sometimes assumed to “prove”the vesicular transport model -- look a thenames of the Golgi stacks used in thisexperiment. However, the experiment can beread backwards as well.

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• The Trans Golgi Network (TGN) is a sortingstation.– A reminder. Up to now, we have been dealing

with “bulk flow”.– Evidence for bulk flow.– As we will see material can leave the TGN by

bulk flow.– However, for the first time in this pathway,

there is sorting; that is, cargo is separatedfrom the bulk flow pathway, 48

• What is secretion?

Fig. 15.28

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Chapter 15. Secretion

• Constitutive secretion.

– Always “on” (at least in most cell types)

– The normal route for membrane lipids andproteins to reach the plasma membrane.

– A continuation of bulk flow that we have seen inprevious steps. 50


• Constitutive secretion.– (movie next slide)– Plasma membrane protein (hooked to GFP)

• Made in ER• Moves through Golgi• Goes to plasma membrane

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• Regulated secretion– The reason for the name.– Generally, cells that undergo regulated

secretion only make one protein for regulatedsecretion.

– Some examples of regulated secretion.– The importance of sorting.– Clathrin coated vesicles are involved.– Aggregation of contents.

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Chapter 15. Endocytosis

• Types of endocytosis.– Pinocytosis– Phagocytosis– Clathrin mediated endocytosis (= Receptor

mediated endocytosis)

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• An example of clathrin mediatedendocytosis: uptake of LDL. (c.f. Fig. 15-39)

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• Molecular defects in clathrin mediatedendocytosis of LDL particles have medicalsignificance.– In some cases LDL receptor cannot bind the

LDL particle.• High circulating LDL in blood, cells starved for

cholesterol.

– In other cases LDL receptor cannot bindclathrin based sorting system.• Symptoms as above.

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• Many other molecules are also taken up byclathrin mediated endocytosis.– Vitamins– Iron

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Chapter 15. Endosomes and Lysosomes

• Endosomes are the default target forendocysosis.

• Viruses can also use this pathway to entercells.

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• Endosomes are sorting stations.– The three pathways from the endosome

• Recycling (like the LDL)• Default: degradation.• Another domain in polarized cells (transcytosis).• See Fig 15-33.

59Fig. 15.33

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• Lysosomes are thesites of intracellulardigestion.

• What is a lysosome?

Fig. 15.34

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• How is material that is fated to bedegraded get loaded into a lysosome.

Fig. 15.35

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• How do the hydrolyases get loaded into theendosome/lysosome?– Hydrolyases get made in the RER, imported into

its lumen and get glycosylated during import.– The oligosaccharides on the hydrolyase get

modified during transit in the ER/Golgi resultingin a mannose-6-phosphate “tag”.

– All proteins with this tag are sorted intoclathrin-coated transport vesicles at the TGN.

– The receptors are recycled between TGN andendosome.

Chapter 15. Overview of Vesicular Transportlewisj/Cell.04/Lectures/Chap15b.6.pdfOverview of Vesicular Transport •Vesicle formation, targeting and fusion are separate events, each

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