Lecture 23: Secretory Pathway & Protein Quality Control

secretory pathways includes proteins that are ________ from the cell or become ________ proteins

secreted; membrane

in addition to secreting proteins, the secretory pathways also inserts and folds ________

integral membrane proteins

the cell is packed with ________

membranes

what type of experiment did Palade use to study the secretory pathway?

Pulse-chase experiment

pulse-chase experiments are good for studying ________ pathways/processes

continuous

what type of radioactive amino acid did the palate pulse-chase experiment use?

leucine

how were labeling amino acids useful for following the secretory pathway?

radioactive aa were incorporated into secretory proteins

palace fixed cells for ________ at various time points

electron microscopy

in the palade pulse-chase experiment, the radioactive grains appear as ________

black dots

immediately after the pulse, the radioactivity was found on top of the ________

rough ER

40minutes after the exposure, the radioactivity was found in ________

vacuoles

the Palade experiment discovered that the pathway of secretory proteins was:

ER → Golgi → vesicles → out of cell

in this table: what do each of the percentages mean?

the amount of radioactivity in each corresponding compartment

the secretory pathway ________ and ________ proteins for secretion

prepares; packages

what is the process by which vesicles release proteins outside the cell

exocytosis

what are the 3 parts of the golgi?

cis, medial, trans

what is the other with which secretory proteins move through the different golgi?

cis → medial → trans

compartments of the secretory pathway are ________ equivalent

topologically

the inside of vesicles are equivalent to the ________ of the cell

outside

the ________ is the central organelle of the secretory pathway

golgi

Othman reconstituted golgi trafficking in ________

cell-free extracts

in rothmans experiments; he creates a mutated cell line that lacked ________ in addition to a wilftype cell line that lacked the ________

glycosyltransferase; protein

without complementation neither the mutant or wild type would express a ________ (Rothman experiment)

glycosylated VSV-G protein

if the cell lines were mixed; you could see the ________ protein ONLY if the protein was ________ via a ________ from mutant to WT cell (Rothman experiment)

glycosylated; transferred; vesicle

in the Rothman experiment; only the wild type ________ golgi was able to complement the glycosylation defect of the mutant cell line

medial

the Rothman experiment allowed them to identify which ________ are needed for ________

proteins; trafficking

membrane traffic begins with ________ and formation of the ________

cargo selection; vesicle

when the vesicle first starts budding off the membrane; it is covered in ________

coat proteins

during vesicle transport, the ________ are removed from the outside

coat proteins

after vesicle transport, the vesicle will ________ on the accepting membrane

dock

after the vesicle docks with the membrane; it will ________ via ________ proteins

fuse; t-SNARE

snare proteins are needed to mediate ________

fusion

how do SNARE proteins mediate fusion?

wind together with proteins on vesicle

are snare proteins only found on the target membrane?

no on vesicle and target membrane

the target membrane SNARE protein is called ________, and the vesicle version is called ________

t-SNARE; v-SNARE

what are three jobs of the ER that help mediate formation of a mature protein?

folding, disulfide formation, and glycosylation

the ER can induce ________ cleavage and the formation of ________ complexes

proteolytic; multimeric

in addition to proteins; the ER plays a large role in ________ and ________ synthesis

lipid; cholesterol

proteins are folded via the ________ effect

hydrophobic

protein folding is due to ________ regions folding to exclude ________ and increase ________

hydrophobic; water; entropy

protein folding is often assisted by ________

chaperones

what does BiP stand for?

binding protein

what does BiP do?

assist protein folding

BiP will bind to ________ regions of a peptide as it is being ________ and threaded into the ________

hydrophobic; translated; ER

BiP proteins also use ________ to help ________ the peptide through the translocase

energy; pull

what is PDI used for?

fixing and making disulfide bonds

why are misfiled proteins bad?

they aggregate and cause disease

what are 3 diseases that are caused by aggregation of misfolded proteins?

Alzheimer’s, Huntington’s, Parkinson’s

what is a disease that is caused by a mutated protein, but NOT caused by aggregation?

cystic fibrosis

what protein is mutated that causes cystic fibrosis?

chlorine channel

what mutation causes CF?

phe deletion

is the Phe deletion that causes CF located near the channel?

no! (its no where near the “active site”)

why does the Phe deletion cause issues in the protein?

slow to fold, cells degrade it

because they are a lack of Cl- channels on the membrane of CF patients, this causes the ________ to be thrown off leading to an excess of ________

osmotic balance; mucus

there is a fine balance between the proper ________ of a protein and the ________ required to do so

folding; length of time

glycosylation is used as ________ for proteins

quality control

sugars are added to ________ proteins, this allows them to bind to ________ on the ER membrane which helps them fold. once the protein is folded, the sugar is ________

unfolds; calnexin; chopped off

where is calnexin located?

ER membrane

what happens when a protein leaves calnexin, but is still not properly folded?

glycosyl transferase adds another sugar on, and cycle repeats

the ER has lots of potential for ________ because it is small and there are many unfolded proteins

aggregation

when there is an increase of aggregation in the ER, this caused ________

ER stress

when the ER is stressed; you want to ________ translation, ________ the number of chaperones, ________ transcription, ________ ER volume, and ________ cell growth

decrease; increase; decrease; increase; stop

cell growth can leads to an ________ in ER stress

increase

during ER stress, you want to decrease the amount of mRNA, specifically for ________ proteins

ER-bound

________ and ________ are key signalers for unfolded protein response (UPR)

IRE1; PERK

what is UPR

unfolded protein response

what kind of proteins are IRE1 and PERK?

kinase sensors

ER stress causes the ________ (general)

unfolded protein response

the unfolded protein response induces actions to help decrease ________

ER stress

IRE1 and PERK sense ________ proteins and will ________ causing a series of ________

unfolded proteins; dimerize; phosphorylations

what is ATF6?

a UPR sensor

ATF6 is normally held in the ________, however, during ER stress it will travel to the ________ where it will be cleaved and become a ________

ER; golgi; TF

PERK phosphorylates a ________ factor

translation initiation factor

which IF does PERK inhibit?

eIF2

the inactive form of eIF2 is ________

phosphorylated

when eIF2 is phosphorylated, ________ will decrease

translation

the inactivation of eIF2 will increase the activity of ________

ATF4

what does ATF4 do?

activate transcription of chaperones

IRE1 is a ________/________ that will splice its target

kinase; endonuclease

IRE1 is activated by binding to ________

unfolded proteins

once IRE1 is activated it ________

ligimers

the ________ activity of IRE1 will cleave the XBP1 mRNA

ribonuclease

XBP1 mRNA is an inactive mRNA because an ________ was left it in

intron

IRE1 cleaves the ________ out of XBP1 mRNA, which activates it for ________

intron; translation

the XBP1 protein is a ________ for the expression of ________

TF; chaperones

in addition to cleaving XBP1 mRNA, IRE1 is able to degrade mRNA located at the ________, which help reduce the load of ________ on the ER

translocon; proteins

which particular mRNA sequence does IRE1 splice and activate?

XBP1

ER-associated degradation targets ________ misfolded proteins for ________

terminally; degradation

what is ERAD?

ER-associated degradation

why does the ER need to transport proteins OUT of the ER for degradation?

no proteosomes in the ER

what is the process called of moving a protein back out of the ER?

reverse translocation

what are 2 ways that a UPR is able to sense a misfolded protein?

exposed hydrophobic regions and unformed disulfides

when the ER stress is too great, what does the cell do?

apoptosis (cell death)

how does type 2 diabetes cause ER stress

increased demand for insulin secretion

how could you induce ER stress in the lab?

inhibit glycosylation

two of the main functions of the Golgi is ________ of glycosyl modifications and ________ of proteins into secretion ________

remodeling; packaging; vesicles

which once of the following proteins does NOT enter the secretory pathway?

SRP

disulfide bonds are common in proteins to be ________

exported (secreted)

what does Hsp 70 do?

heat shock chaperones

what does calreticulin do?

assist with protein folding/chaperoning