Exam 3: Ch 14 Secretory Pathway & Vesicle Budding

Question 1

the secretory pathway carries both…

Answer 1

soluble and membrane proteins from the ER to their destination on the cell surface or the lysosome

Question 2

soluble proteins delivered to the lysosome include

Answer 2

lysosomal digestive enzymes like…

proteases, phosphatases, lipases

Question 3

endocytic pathway

Answer 3

used to take up substances from the cell surface and move them into the interior of the cell

ingest nutrients that are too big to be transported (ex. LDL and iron)

remove receptor proteins from the cell surface to down-regulate their activity

Question 4

unifying principle that governs all protein trafficking in secretory and endocytic pathways

Answer 4

transport of membrane and soluble proteins from 1 membrane bound compartment to the next is mediated by transport vesicles

these vesicles collect “cargo” proteins in buds, then deliver them by fusing with the target membrane

Question 5

when transport vesicles bud from one membrane and fuse to the next, does the same face of the membrane remain oriented toward the cytosol?

Answer 5

yes

therefore, the protein being transported retains its original orientation

Question 6

stage 1 of secretory pathway

Answer 6

proteins are translocated to the ER where they properly fold and receive modifications like N/O-linked carbohydrates and disulfide bonds

Question 7

stage 2 of the secretory pathway

Answer 7

the properly folded proteins are transported via anterograde vesicles to the Golgi that fuse to form cis-golgi

retrograde vesicles moves proteins back to the ER

cis-golgi –> medial-golgi –> trans-goli (cisternal maturation)

retrograde vesicles move golgi proteins from trans to cis

Question 8

trans-golgi network (TGN)

Answer 8

major branch point in secretory pathway

here, proteins are loaded into different vesicles and trafficked to different destinations

Question 9

destinations of proteins from the TGN

Answer 9

plasma membrane –> secretion

storage

lysosome

Question 10

exocytosis

Answer 10

process where a vesicle to and fuses with a plasma membrane and releases its contents

Question 11

late endosome

Answer 11

a compartment where secretory proteins destined for the lysosome end up first

Question 12

where are secretory proteins never released

Answer 12

cytosol

they are always associated with some kind of membrane bound intermediate

Question 13

combining pulse-chase labeling and autoradiography (radiolabeled aa)

Answer 13

after injection of labeled aa in pancreas of hamsters, they were killed and pancreatic cells visualized to detect the location of labeled protein

Question 14

vesicular stomatitis virus procedure (VSV)

Answer 14

temperature sensitive mutant G protein from VSV transfected into cells that then produce G protein on rough ER

at restrictive temp of 40 degrees C, no transport occurs

at permissive temp of 32 degrees C, transport occurs through secretory pathway to cell surface

Question 15

microscopy of GFP labeled VSV G protein

Answer 15

hybrid gene of GFP + VSVG

when cells transcribe gene, fluorescent microscope used to observe

protein moves to golgi, then cell surface

Question 16

endoglycosidase D

Answer 16

carbohydrate cleaving enzyme

distinguish glycosylated proteins in ER (resistant to cleavage) from those that enter cis-Golgi

deglycosylated protein moves faster on SDS gel

Question 17

temperature (hot or cold) sensitive secretion (sec) mutants in coding region

Answer 17

yeast mutants that secrete proteins at a permissive temp, but not at a higher nonpermissive temp

at nonpermissive temp, see which proteins build up and where

Question 18

what is budding of vesicles driven by

Answer 18

combination of soluble protein complexes onto the membrane to form a protein vesicle coat

Question 19

cytosolic portions of integral membrane proteins interact with the vesicle coat to…

Answer 19

uptake appropriate cargo proteins into forming vesicle

causes curvature

Question 20

v-SNARE

Answer 20

vesicle SNARE proteins in protein coat accessible in uncoated vesicle (when pinched off)

binds to t-SNARE in target memb. to bring the membranes close together, allowing bilayers to fuse

Question 21

t-SNARE

Answer 21

found on target membrane (target SNARE)

v-SNARE binds and allows membranes to fuse

Question 22

classic coat proteins

Answer 22

COPII vesicles transport proteins from ER to golgi (anterograde)

COPI vesicles transport proteins retrograde between golgi compartments, and from cis-golgi to ER

clathrin vesicles transport proteins from plasma membrane and trans-golgi to endosomes (ex. cholesterol)

Question 23

is the formation of vesicles energy dependent?

Answer 23

yes

Question 24

why is movement in golgi retrograde

Answer 24

vesicles stay stationary as golgi matures

Question 25

do some vesicles move on microtubules?

Answer 25

yes

Question 26

GTPase switch proteins control...

Answer 26

assembly of vesicle coat proteins (ex. ARF protein, Sar1) Sar1-ATP helps coat proteins bind, hydrolysis to GDP causes release

Question 27

Sar1 / ARF protein mutants that cant hydrolyze GTP-GDP

Answer 27

protein coats form, but never disassemble

Question 28

how does the vesicle coat select the right cargo proteins?

Answer 28

cargo proteins have sorting signals

Question 29

___ GTPases control docking of vesicles on target membranes

Answer 29

Rab has an anchor that allows tethering to vesicle memb. (Rab-GTP) Rab-GTP interacts with a Rab effector in target membrane to dock vesicle then Rab-GTP --> Rab-GDP = release

Question 30

VAMP (v-SNARE) and syntaxin (t-SNARE) binding mechanism in secreted protein exocytosis

Answer 30

VAMP incorporated into secretory vesicles as they bud from trans-Golgi when protein coat disassembles, VAMP exposed, binds to syntaxin after Rab-GTP tethering to memb. forms SNARE complex

Question 31

dissociation of SNARE complexes after membrane fusion is driven by ___ hydrolysis

Answer 31

ATP

Question 32

anterograde transport from ER to golgi uses which vesicle type

Answer 32

COPII vesicles contain proteins destined to golgi, cell surface, or lysosomes, and v-SNARES

Question 33

retrograde transport from cis-Golgi to ER uses which vesicle type

Answer 33

COPI retrieves v-SNARES, membrane, and missorted ER proteins

Question 34

KDEL sorting signal

Answer 34

found on proteins destined to ER from golgi proteins carried by COPI vesicles

Question 35

golgi processes post-translational modifications to what proteins

Answer 35

glycoproteins contains enzymes like glycosidases and glycosyltransferases (add n/o-linked carbohydrates) add mannose-6-phosphate in cis-golgi

Question 36

vesicles that bud from trans-golgi to final destinations have two layers

Answer 36

outer layer of clathrin (trimeric) inner layer of adapter protein (AP) complexes, 3 kinds

Question 37

AP proteins

Answer 37

determine which cargo proteins are included in vesicle bud bind cytosolic face of membrane proteins

Question 38

trans golgi proteins going to the lysosome through late endosome contain

Answer 38

clathrin and AP1

Question 39

trans golgi proteins going to lysosome by bypassing late endosome contain

Answer 39

AP3 MAYBE clathrin

Question 40

______ is required for pinching off clathrin vesicles

Answer 40

Dynamin, a cytosolic protein forms around neck of bud and hydrolyzes GTP-->GDP

Question 41

do COPI and COPII need dynamic to pinch off?

Answer 41

no

Question 42

mannose 6-phosphate residues target proteins to ______

Answer 42

lysosomes trans-golgi has mannose 6-phosphate receptors

Question 43

some mannose 6-phosphate glycoproteins are ___-proteins initially synthesized as...

Answer 43

pro-proteins pre-pro-proteins like lysosomal proteases

Question 44

there are receptors for M6P proteins in the ____ golgi

Answer 44

trans bind to clathrin/AP1 vesicles --> late endosome --> lysosome

Question 45

lysosomal storage disease (genetic)

Answer 45

degradative enzyme is defective in active site of M6P or in sorting/transport process absence of one or more lysosomal enzymes results in buildup of glygolipids in lysosomes

Question 46

secretory cells like pancreatic beta cells (insulin) use 2 types of secretory vesicle

Answer 46

regulated transport vesicles unregulated transport vesicles (constitutive)

Question 47

proprotein

Answer 47

long lived inactive precursor proteins need further proteolytic processing to generate mature active proteins

Question 48

which proteins undergo proteolytic processing after leaving trans-golgi

Answer 48

lysosomal pro-enzymes (happens in late endosome) pro-insulin (happens in vesicles after leaving trans golgi)

Question 49

how do epithelial cells get to either the apical or basolateral region

Answer 49

sorting pathways in the trans golgi different transport vesicles to apical or basolateral surfaces

Question 50

transcytosis

Answer 50

when epithelial cells destined for the apical side of the membrane move across the cell and fuse