Exam 3: Ch 14 Secretory Pathway & Vesicle Budding Flashcards
the secretory pathway carries both…
soluble and membrane proteins from the ER to their destination on the cell surface or the lysosome
soluble proteins delivered to the lysosome include
lysosomal digestive enzymes like…
proteases, phosphatases, lipases
endocytic pathway
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
unifying principle that governs all protein trafficking in secretory and endocytic pathways
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
when transport vesicles bud from one membrane and fuse to the next, does the same face of the membrane remain oriented toward the cytosol?
yes
therefore, the protein being transported retains its original orientation
stage 1 of secretory pathway
proteins are translocated to the ER where they properly fold and receive modifications like N/O-linked carbohydrates and disulfide bonds
stage 2 of the secretory pathway
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
trans-golgi network (TGN)
major branch point in secretory pathway
here, proteins are loaded into different vesicles and trafficked to different destinations
destinations of proteins from the TGN
plasma membrane –> secretion
storage
lysosome
exocytosis
process where a vesicle to and fuses with a plasma membrane and releases its contents
late endosome
a compartment where secretory proteins destined for the lysosome end up first
where are secretory proteins never released
cytosol
they are always associated with some kind of membrane bound intermediate
combining pulse-chase labeling and autoradiography (radiolabeled aa)
after injection of labeled aa in pancreas of hamsters, they were killed and pancreatic cells visualized to detect the location of labeled protein
vesicular stomatitis virus procedure (VSV)
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
microscopy of GFP labeled VSV G protein
hybrid gene of GFP + VSVG
when cells transcribe gene, fluorescent microscope used to observe
protein moves to golgi, then cell surface
endoglycosidase D
carbohydrate cleaving enzyme
distinguish glycosylated proteins in ER (resistant to cleavage) from those that enter cis-Golgi
deglycosylated protein moves faster on SDS gel
temperature (hot or cold) sensitive secretion (sec) mutants in coding region
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
what is budding of vesicles driven by
combination of soluble protein complexes onto the membrane to form a protein vesicle coat
cytosolic portions of integral membrane proteins interact with the vesicle coat to…
uptake appropriate cargo proteins into forming vesicle
causes curvature
v-SNARE
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
t-SNARE
found on target membrane (target SNARE)
v-SNARE binds and allows membranes to fuse
classic coat proteins
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)
is the formation of vesicles energy dependent?
yes
why is movement in golgi retrograde
vesicles stay stationary as golgi matures
do some vesicles move on microtubules?
yes
GTPase switch proteins control…
assembly of vesicle coat proteins (ex. ARF protein, Sar1)
Sar1-ATP helps coat proteins bind, hydrolysis to GDP causes release
Sar1 / ARF protein mutants that cant hydrolyze GTP-GDP
protein coats form, but never disassemble
how does the vesicle coat select the right cargo proteins?
cargo proteins have sorting signals
___ GTPases control docking of vesicles on target membranes
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
VAMP (v-SNARE) and syntaxin (t-SNARE) binding mechanism in secreted protein exocytosis
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
dissociation of SNARE complexes after membrane fusion is driven by ___ hydrolysis
ATP
anterograde transport from ER to golgi uses which vesicle type
COPII
vesicles contain proteins destined to golgi, cell surface, or lysosomes, and v-SNARES
retrograde transport from cis-Golgi to ER uses which vesicle type
COPI
retrieves v-SNARES, membrane, and missorted ER proteins
KDEL sorting signal
found on proteins destined to ER from golgi
proteins carried by COPI vesicles
golgi processes post-translational modifications to what proteins
glycoproteins
contains enzymes like glycosidases and glycosyltransferases (add n/o-linked carbohydrates)
add mannose-6-phosphate in cis-golgi
vesicles that bud from trans-golgi to final destinations have two layers
outer layer of clathrin (trimeric)
inner layer of adapter protein (AP) complexes, 3 kinds
AP proteins
determine which cargo proteins are included in vesicle bud
bind cytosolic face of membrane proteins
trans golgi proteins going to the lysosome through late endosome contain
clathrin and AP1
trans golgi proteins going to lysosome by bypassing late endosome contain
AP3
MAYBE clathrin
______ is required for pinching off clathrin vesicles
Dynamin, a cytosolic protein
forms around neck of bud and hydrolyzes GTP–>GDP
do COPI and COPII need dynamic to pinch off?
no
mannose 6-phosphate residues target proteins to ______
lysosomes
trans-golgi has mannose 6-phosphate receptors
some mannose 6-phosphate glycoproteins are ___-proteins initially synthesized as…
pro-proteins
pre-pro-proteins like lysosomal proteases
there are receptors for M6P proteins in the ____ golgi
trans
bind to clathrin/AP1 vesicles –> late endosome –> lysosome
lysosomal storage disease (genetic)
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
secretory cells like pancreatic beta cells (insulin) use 2 types of secretory vesicle
regulated transport vesicles
unregulated transport vesicles (constitutive)
proprotein
long lived inactive precursor proteins
need further proteolytic processing to generate mature active proteins
which proteins undergo proteolytic processing after leaving trans-golgi
lysosomal pro-enzymes (happens in late endosome)
pro-insulin (happens in vesicles after leaving trans golgi)
how do epithelial cells get to either the apical or basolateral region
sorting pathways in the trans golgi
different transport vesicles to apical or basolateral surfaces
transcytosis
when epithelial cells destined for the apical side of the membrane move across the cell and fuse
