LEC35: Secretory Pathway and Endocytosis (Part D: Endocytosis, Part E: Mechanisms of Vesicular Transport)

Question 1

basic function of smooth ER?

rough ER?

golgi complex?

Answer 1

smooth ER: biogenesis of lipids for membranes

rough ER: biosynthesis of protein

golgi: sorting, according to destination, of protein

Question 2

regulated vs constituitive secretion purpose?

Answer 2

regulated secretion: need signal to get taken to plasma membrane

constituitive secretion: housekeeping molecules that’re constantly made, in different cell types

Question 3

what is endocytosis?

Answer 3

process of how materals are moved from the outside to inside of the cell

occurs by invagination of the plasma membrane via endocytic pathway

2 processes do this: pinocytosis and phagocytosis

Question 4

what is phagocytosis? what sized molecules undergo this?

Answer 4

ingestion of molecules >0.5 microns

occurs by professional scavenging cells like macrophages which can invaginate large areas of plasma membrane to encompass pathogenic bacteria

recycles things in blood and foriegn bodies, breaks foreign bodies down to constituent parts

Question 5

what endocytic pathway do red blood cells undergo?

Answer 5

phagocytosis

up to 10¹¹RBC are recycled daily by phagocytosis

Question 6

what is pinocytosis? what sized molecules undergo this?

Answer 6

cells that invaginate and form smaller vesicles

helps **recycle membranes and transport materials into cells **

cell membrane invagination occurs constituitively in very small vesicles during pinocytosis

ingests materials < 0.5 microns in size

Question 7

what type of process is receptor mediated endocytosis?

Answer 7

a type of pinocytosis

Question 8

what are 2 examples of receptor mediated endocytosis?

Answer 8

1) LDL is endocytosed by its receptor
2) transferrin is endocytosed when binds to its receptor
* receptor mediated endocytosis is a form of pinocytosis*

Question 9

what is LDL

Answer 9

low density lipoprotein, carries cholesterol in the blood

structure is large molecule of:

1) 1,500 molecules of cholesterol esters in the middle,
2) 1 huge protein, ApoB100, and
3) phospholipid/cholester coat w/ cholesterol on it

Question 10

describe the process of receptor mediated endocytosis between LDL and the LDL receptor

Answer 10

1) LDL receptor is on plasma membrane

Apo-B protein of LDL binds to LDL receptor; this internalizes LDL w/ its receptor

2) LDL receptor w/ hundreds of LDL particle/receptor complexes enter cell, fuse w/ the endosome
3) b/c endosome pH=5.5, LDL is released from its receptor into endosome

LDL receptor is recycled back to plasma membrane

4) endosome and lysosome fuse, LDL transfers into lysosome
5) in lysosome, lysosomal enzymes attack LDL, break it down, return free cholesterol back into the cell

Question 11

describe process of how Fe3+ moves into cells

Answer 11

via receptor mediated endocytosis, like w/ LDL

1) Fe3+ binds to transferrin, a protein in the blood
2) transferrin receptors on cell membrane bind transferrin-Fe3+
3) these receptor-transferrin-Fe3+ are bound into vesicles that fuse w/ the endosome
4) endosome pH = 5.5 does not release transferrin protein; only releases the bound Fe3+

Fe3+ thus is reduced from Fe3+ to Fe2+

5) Fe2+ is exported out of the endosome by a divalent methyl transporter
6) Apo-transferrin, transferrin w/o Fe3+, is recycled back to the membrane

transferrin w/o Fe3+ fuses w/ the membrane, is released into the extracellular space, returns to blood to pick up more iron

Question 12

what is autophagy?

Answer 12

how intracellular components are delivered to lysosome for destruction and recycling

Question 13

describe process of autophagy

Answer 13

1) organelle/protein aggregate is engulfed by a autophagosome, double membrane structure
2) autophagosome fuses with lysosome to form **autophagolysosome **

Question 14

what is the relationship between autophagy and age?

Answer 14

direct relationship: if have better autophagy as you age, you live longer (shown in worm, fly, yeast)

Question 15

how often does autophagy occur?

Answer 15

constant process

Question 16

what is this?

Answer 16

EM of LDL receptor w/ LDL particles going through membrane invagination, encircling by vesicle, vesicle to membrane, and then pinched off

Question 17

why does vesicle budding happen?

Answer 17

in response to need for cargo to be transported from 1 compartment to another in secretory pathway

e.g. ER to Golgi, Golgi to Lysosome, etc

Question 18

what process is integrated alongside budding process?

Answer 18

cargo selection via “coat” proteins that bend the membrane into vesicles

Question 19

what are different types of coat proteins? what type of vesicles does each coat?

Answer 19

1) clathrin: vesicles from trans-Golgi network to plasma membrane and to endosomes/lysosomes
2) COPI: vesicles from cis Golgi to ER
3) COPII: vesicles from ER to plasma membrane

Question 20

when does vesicle’s coating form?

Answer 20

alongside formation of vesicles -

when receptor binds cargo, molecule of clathrin or COPI or COPII forms also

Question 21

what is structure of clathrin molecule?

Answer 21

heavy and light chain

has polymerizing units, triskelions that interact w/ each other, polymerize into shape (see image) which drags membrane into a curve form, allows vesicles to form

properties of polymerization means vesicles have uniform 30 nm size

Question 22

what processes are integrated w/ vesicle budding?

Answer 22

cargo selection via proteins that bind to both cargo itself and to ‘coat’ proteins that help beind membrane into vesicles

coat proteins = clathrin, COPI, COPII

Question 23

describe process of when/how coat proteins bind to vesicles

Answer 23

1) in TGN, when receptor and cargo bind, receptor binds to Adaptin AP1 molecule
2) Adaptin binds clathrin

or

1) in plasma membrane, when receptor and cargo bind, Adaptin is AP2
2) AP2 binds clathrin

Question 24

how do clathrin molecules aid w/ vesiclular formation? describe process of vesicle budding via clathrin increase

Answer 24

1) when there are more receptors on membrane, that means there are more clathrin molecules prsent
2) when clathrins are close to each other, they begin to polymerize
3) as they polymerize, they pull the membrane away into a vesicle
4) vesicle sits on the membraen until acted on by dynamin, protein that uses GTP hydrolysis to pinch vesicle off the membrane

Question 25

what is dynamin? how would its action be inhibited?

Answer 25

protein that cuts clathrin-coated vesicle from the plasma membrane

works by GTP hydrolysis

so if you inhibit the GTPase, dynamin cannot complete action of pinching off the plasma membrane, even if clathrin coat has been made

Question 26

what happens to clathrin coat when vesicle buds and pinches off the membrane? why does this matter?

Answer 26

coat falls off, disassembles

important b/c targeting mechanism is on the vesicle so if coat is covering vesicle then targeting doesn’t happen

Question 27

what are COPI and II coats a part of?

Answer 27

ER retrieval pathway and movement of vesicles within golgi itself

Question 28

what are the general functions of RAB and SNARE proteins?

Answer 28

target vesicles specifically to different membranes depending on the cargo they carry

Question 29

what does RAB protein do? describe mechanism of action

Answer 29

protein responsible for executing targeting mechanism for vesicles

1) Rab binds to vesicle
2) Rab binds to long tethering protein called Rab effector, long fibrous protein tethered to target membrane
3) Rab effector bound to Rab protein brings vesicle into close apposition w/ correct membrane
4) fusion of membranes occurs via SNARE proteins

Question 30

what do SNARE proteins do?

Answer 30

responsible for vesicle fusion

overcomes enegetically unfavorable fusion by interactions of V-SNARE proteins on the vesicle and T-SNARE proteins on target membrane

Question 31

why is vesicle fusion energetically unfavorable?

Answer 31

the hydrphilic head groups and their associated water molecules represent a barrier to mixing of hydrophobic hydrocarbons

Question 32

how does SNARE fusion occur after Rab targeting?

Answer 32

V-snare and T-snare get close to each other

interact in a coiled coil around each other

when snare proteins pull from either side, they pull vesicle and membraen close to each other

interaction of SNARE proteins squeezes out all the water moelcules and reducing thermodynamic barriers to lipid mixing

after fusion, SNARE proteins are disassembled w/ help of a specific chaperone, then recyled back to respective membrane systems

Question 33

what is SNARE system analogous to?

Answer 33

how viruses get into cells by squeezing tighter and tighter until fusion of membraens occurs

Question 34

what is NSF?

Answer 34

chaperone protein that undoes SNARE-v and SNARE-t winding/binding