Lecture 8

Front 1

Vesicular transport

Back 1

transport between ER, golgi and various endosomes(between topologically equivalent spaces)

never cross membrane

Front 2

Vesicular transport (3 pathways)

Back 2

1. biosynthetic-secretory

2. endocytosis

3. retrieval pathways

Front 3

biosynthetic secretory pathway

Back 3

new proteins, carbs and lipids delivered to plasma membrane or extracellular space

leads outward from the ER to the Golgi and cell surface, with a side route leading to lysosomes

Front 4

endocytosis

Back 4

molecules are ingested via endocytotic vesicles derived from plasma membrane and delivered to endosomes or lysosomes

Front 5

retrieval pathways

Back 5

return memb. and select proteins back to compartments of origin to be re-used

Front 6

vesicle coat

Back 6

cage of proteins on the cytosolic surface of the vesicle

type of coat used will depend on what cargo is being transported and where it is going

Front 7

Functions of vesicle coats

Back 7

1. collect and concentrate specific memb. and soluble cargo molec. for transport at a patch of memb.

2. drives formation of round-shaped vesicles

Front 8

Types of Vesicle coats

Back 8

1. Clathrin

coats vesicles moving away from the plasma memb. , and bw golgi and endosomes

2. COPI

coats vesicles bw. golgi compartments

3. COPII

coats vesicles moving from ER-> Golgi

Front 9

Back 9

Clathrin-coated vesicles

Front 10

mechanism of clathrin-coated vesicles

Back 10

forms triskelion

forms a cage around a budding vesicle (NO NRG)

cargo receptors: transmembrane proteins that capture soluble cargo inside the budding vesicle

adaptor proteins: bind receptors or transmembrane proteins to clathrin

dynamin: cytoplasmic GTPase that assembles at the neck of each bud

-pinches off vesicle

-requires GTP hydrolysis for NRG

Front 11

coat removal in Clathrin coated vesicles

Back 11

once vesicle is formed, coat removal need NRG

Hsp70 chaperone proteins use the energy of ATP hydrolysis to peel off coat

Front 12

what determines where and when a vesicle will bud?

Back 12

1. lipid markers

2. coat recruitment GTPases

Front 13

Lipid Markers

Back 13

Phosphotidylinositol (PI) with extra phosphates on head group -> PIP

phosphorylation and de-phosphorylation of PI head groups can produce a variety of PIP species

Front 14

different organelle have different ____ compositions

different adaptor proteins bind to specific ___markers

Back 14

PIP

Front 15

Back 15

interconversion of PI head groups by enzymes (e.g. kinases, phosphatases) can regulate where a vesicle will form

Front 16

coat-recruitment GTPases

Back 16

GTPases = GTP binding proteins

active=bound to GTP; inactive=bound to GDP

Front 17

Sar1 protein

Back 17

functions in the formation of COPII vesicles at the ER membrane

Front 18

Vesicle formation via Coat-recruitment GTPase

Back 18

1. inactive Sar1-GDP in cytosol

2. can be activated by ER memb. GEF which mediates the xchange of GDP-> GTP

3. Sar1-GTP now able to bind to the ER memb.

4. Sar1-GTP recruits adaptor prtn. (sec23,24) for budding initiation

5.incr. in Sar1-GTP activation;incr. adaptor prtns. and COPII molec. recruited to from vesicle

6. once formed, memb. fuse to pinch off the vesicle

Front 19

once vesicle is _________ the coat is ________

Back 19

once the vesicle is formed, the coat is removed

Front 20

In COPII vesicles, _____ hydrolyzes bound GTP-------->GDP

This causes what to happen to the coat??

Back 20

for COPII-coated vesicles, SAR1 hydrolyzes bound GTP to GDP

causes the coat to come off

Front 21

how do vesicles recognize and fuse with the correct target?

Back 21

1. Rab proteins

2. Snare proteins

Front 22

Rab proteins, what are they?

Back 22

GTPase proteins that serve as identity markers

Front 23

what does interaction b/w active Rab proteins on vesicle memb. and Rab effector proteins on the target memb. do ???

Back 23

-tethers vesicle close to the target, allows for fusion

-ensures correct target direction

Front 24

Back 24

Tethering of a transport vesicle to a target membrane.

Front 25

SNARE proteins

Back 25

mediate memb. fusion

2 types:

v-SNARE: on vesicular memb.

t-SNARE: on target memb.

once tethered by Rabs, v+t SNAREs wrap around each other to lock memb. together + promote fusion

Front 26

SNARE protein fusion mediation

Back 26

1. v and t SNARES recognize, and wrap around each other

2. interactions lock 2 memb. into close approximation + squeezes out H2O

3.memb. lipids then flow into each other to fuse memb. together

spontaneous

need ATP to seperate SNAREs

Front 27

Budding of vesicles requires what?

Fusion of vesicles requires?

Back 27

dynamin for budding

SNAREs for fusion

Front 28

Golgi Apparatus Function

Back 28

1) acts as a sorting and transfer station for products of the ER

2) site of carbohydrate synthesis

Front 29

Transport b/w ER and Golgi

Back 29

COPII-coated vesicles at ER exit sites:

1.bind to: (w/ exit signals)

cargo receptors -soluble proteins

memb. prtns. -adaptor proteins within the COPII coat

unfolded prtns bound by chaperone prtns and retained within the ER

most resident ER proteins do not leave the ER; but some accidentally enter the vesicle by bulk flow

Front 30

Back 30

The recruitment of membrane and soluble cargo molecules into ER transport vesicles

Front 31

Once vesicles have budded from the ER, what happens to their COPII coat?

Back 31

They shed their COPII coat!

Front 32

After COPII coat shedded,what do the vesicles do?

Back 32

they fuse together into a vesicular tubular cluster, which moves towards the Golgi on microtubules

as the clusters move towards the Golgi, they bud COPI-coated vesicles containing:

-cargo receptors

-SNAREs

-escaped resident ER proteins

Front 33

How do COPI-coated vesicles return to the ER?

Back 33

via the retrieval pathway

Front 34

Back 34

Vesicular tubular clusters:

Vesicular tubular clusters move along microtubules to carry proteins from the ER to the Golgi. COPI-coated vesicles mediate the budding of vesicles that return to the ER from these clusters (and from the Golgi apparatus)

Front 35

retrieval pathway to the ER

Back 35

ER retrieval sequences!!

ex. resident ER prtns

posses a KDEL sequence=4 a.a.

KDEL receptors: bind KDEL sequences on cargo within the vesicular tubular cluster or Golgi and COPI coat proteins

Front 36

Back 36

The KDEL receptor present in both vesicular tubular clusters and the Golgi apparatus captures the soluble ER resident proteins and carries them in COPI-coated transport vesicles back to the ER. (Recall that the COPI coated vesicles shed their coats as soon as they are formed.) Upon binding its ligands in the tubular cluster or Golgi, the KDEL receptor may change conformation, so as to facilitate its recruitment into budding COPI-coated vesicles.

Front 37

how do KDEL receptors get back to the Golgi from the ER?

Back 37

they are packaged in COPII-coated vesicles and carried back to the Golgi

Front 38

What are KDEL sequences??

Back 38

target peptide sequence which keeps a protein from being secreted from ER

Proteins can only leave the ER after this sequence has been cleaved off.

Front 39

Oligosaccharide Processing in the Golgi

Back 39

sugars are further trimmed and more are added or modified in the Golgi

-occurs in a highly ordered sequence from cis to trans faces, with different processes occurring in different compartments

-enzymes that carry out these processes are localized to different compartments

Front 40

Back 40

Oligosaccharide processing in Golgi compartments.

Front 41

- Oligosaccharide Processing in the Golgi: Function

Back 41

1) assists with correct protein folding in the ER

2) helps glycoproteins resist proteolytic degradation

3) aids in cell-to-cell recognition