ODSC 511C: Protein Localization, Trafficking and Vesicle Transport

can mitochondrial DNA replicate by itself?

yes

which organelle has the main function of "matrix, highway, metabolic pathway, protein synthesis"

cytosol

which organelle has the main function of "storing, replicating, transcribing genetic info"

nucleus

which organelle has the main function of "synthesizing lipids, synthesizing proteins (vesicles and membrane)

endoplasmic reticulum

which organelle has the main function of "modifying, storing and packaging"

golgi apparatus

which organelle has the main function of "ATP synthesis"

mitochondria

which organelle has the main function of "sorting endocytosed material"

endosome

which organelle has the main function of "intracellular degradation"

lysosome

which organelle has the main function of "oxidizing toxic molecules"

peroxisome

what is "a short conserved sequence pattern associated with distinct functions of a protein or DNA."

motif

what are the 3 methods to identify protein targeting sequences?

motif finding

loss of function assay

gain of function assay

what guides the transport and localization of proteins?

specific sequences

what are 3 attributes of protein targeting sequences

consensus motif (a single sequence where only one base or amino acid residue is permitted at each position.)

modular

the position in the sequence matters

what is the destination for a protein with a KR cluster motif? (Ex. PKKKRKV (SV40-Tantigen) or PAAKRVKLD (c-Myc))

nucleus

Proteins with KR motifs that are destined for the nucleus are transported folded or unfolded?

folded

what is the destination for a protein with the following motif:

N-terminal 10-70 AA alternating hydrophobic/positive charge?

( EX. MLSRQSIRFFKPATRTLCSSRYLL; S. cerevisiae cytochome c oxidase)

mitochondria

proteins destined for the mitochondria with N terminal alternating hydrophobic and positive charge aa's are transported folded or unfolded?

unfolded

what is the destination of a protein with the motif of N-terminal hydrophobic clusters (15-30)?

(MMSFVSLLLVGILFHATQA; boving alpha lactalbumin)

endoplasmic reticulum

a protein with the motif of a C-terminal KDEL is destined where?

ER retention

what is the destination of a protein with a motif of Non-N terminal Hydrophobic helix (~20aa)

(ex. ALSIVLPIVLLVFLCLGVFLLW; LDL receptor)

transmembrane

what is the destination of a protein with the motif of a -SKL C terminal?

peroxisomes (PTS1)

what is the motif of proteins destined for lysosomes?

vesicles

where is protein targeting decided?

in the cytosol

what are the two types of transport for protein targeting?

post translational

co-translational

what are the destinations for proteins who are post translationally transported?

mitochondria, chloropolasts, peroxisome, nucleus

all proteins who are co-translationally transported first go through what organelle

endoplasmic reticulum

what are the destinations for proteins who are co-translationally transported?

cell exterior (secretion)

plasma membrane

lysosome

other parts of the endomembrane system

proteins that are imported into the nucleus go through what

the nuclear pore complex

what proteins undergo nuclear import?

folded proteins with NLS extended region

what motif does a protein have to have to undergo nuclear import?

classical motif (KR clusters)

during nuclear import, the KR clusters can also be referred to as

nuclear localization signals

which recycle importin (Carrier protein) unloads cargo during nuclear import?

RAN-GTP

what happens when RANG GTP is hydrolyzed during nuclear importn?

RAN-GDP dissociates from the importin and exists the nucleus

99% of mitochondrial protein is ___ encoded

nuclear encoded

what do you need for mitochondrial protein import?

inner and outer mitochondrial protein translocators (mtHSP70)

what drives translocation aka the inner and outer mitochondrial protein translocators coming together?

ATP

what is the motif for mitochondrial protein import?

N terminal alternative hydrophibic/basic aa's

are the precursor proteins folded or unfolded during mitochondrial protein import?

unfolded

what protein provides the energy for peroxisome protein import?

ubiquitin

peroxisome protein import is similar to what other import?

nuclear

C terminal PTS1 on folded cargo protein is required for what type of protein import?

peroxisome

what is the motif for co-translational transport (ER, vesicle and secretory pathway)

N hydrophobic clusters

during co translational transport: ER, vesicles and secretory pathway

a finished protein is released into the ER when

signal peptidase cleaves the signal peptide

what recycles the signal recognition particle?

GTP hydrolysis

with membrane bound proteins, the start/stop transfer sequences are hydrophobic or hydrophilic?

hydrophobic

what are the two types of membrane bound protein transfers?

single pass TM protein

double pass TM protein

what is the function of chaperon proteins (HSP and PDI)

help to fold the proteins

identify mis-folded proteins and chaperone them to the cytosol for degradation

what is the function of oligosaccharide protein transferase (OST) in ER protein trafficking?

bring proteins to teh glycosylation site

how are proteins transported out of the endoplasmic reticulum?

via vesicles to the golgi

as a misfolded protein leaves the ER for the cytosol, what is added so that it will be degraded?

a ubiquitin tag

how do proteins move between stacks in the golgi?

via vesicles

in the golgi, proteins are sorted into what two pathways?

endocytic or secretory

the exocytic pathways have what type of coats?

clathrin + adaptin 1

COP proteins

the endocytic pathways have what types of coats?

clathrin + adaptin 2

what do coat proteins need in order to line up on the membrane to form vesicles?

they need to be energized (hydrolysis of GTP)

what are SNARE proteins?

molecular motors that drive the fusion of two membranes

during vesicle formation, the soluble cargo protein attaches to what for initiation? (2)

transmembrane cargo protein receptor

v SNAREs

dynein moves in what direction?

retrograde

+ to -

kineasin moves in what direction?

anterograde

- to +

the twisting of what SNARE's drives membrane fusion during vesicle delivery of a vesicle

v-SNARE and t-SNARE

what recruits the vesicle during exocytosis?

tethering factor (receptors for vesicles)

what helps to "reload" GTP onto Rab GTPase?

GEF- guanine nucleotide exchange factor

what is the destination code for vesicle during delivery?

Rab protein and tethering factor

is t-SNARE on the membrane of the donor or acceptor?

acceptor

is v-SNARE on the membrane of the donor or acceptor?

donor and then is incorporated into the vesicle

vesicles leave the trans golgi network in one of which two pathways?

constitutive

regulated

vesicles that leave the TGN in the constitutive pathway contain what

newly synthesized plasma membrane proteins and lipids

vesicles that leave the TGN in the regulated pathway contain what

secretory proteins

what is the signal for regulated secretion?

hormone or neurotransmitter

phagocytosis occurs via

phagosomes (>250nM)

pinocytosis occurs via

small vesicles

receptor mediated endocytosis increases the ___ and ___ of target uptake

specificity and efficiency

what unloads the cargo during receptor mediated endocytosis?

acidic endosome

where does complete digestion occur via receptor mediated endocytosis?

lysosome

what type of receptors are necessary during receptor mediated endocytosis?

LDL

during receptor mediated endocytosis, an uncoated vesicle becomes an

early endosome

early endosomes are very

acidic

what do early endosomes become?

lysosomes

phagocytosis is usually ___ mediated

receptor mediated

what are the pseudopods of phagocytes made of

actin

phagosomes fuse with what to digest content

lysosomes

phagosomes use what to degrade contents?

proteases and lipases

what is the pH of lysosomes?

5

waht maintains the pH of lysosomes?

H+ ATPase

what do lysosomes transport?

metabolites

what are the acid hydrolases stored in lysosomes?

nuclease

protease

lipase

glycosidase

phosphatase

phospholipase

sulfatase

A ___ determines which pathway to continue translation (post vs co translational transport)

signal peptide

nuclear imports have more than ___ types of protein

30

what is the translocation motor in mitochondrial protein import?

mt HSP70

mt HSP70 requires ___ to function as translocation motor

ATP

what are the 4 steps of degradation of ER misfolded proteins?

1. substrate recognition

2. retro-translocation

3. poly ubiquitination

4. proteasomal degradation

vesicle or v-SNAREs, incorporated into the membranes of transport vesicles during

budding

what SNARE protein associated with the nerve terminal membranes, mediate exocytosis

target or t-SNAREs

during docking (vesicle transport), v- and t-SNAREs assemble into a ____ which promotes fusion of the vesicle and acceptor lipid bilayers.

four-helix bundle/ "trans-SNARE complex"

plasma proteins leave the golgi and go into the extracellular space via ___ secretion

constitutive

neurotransmitters and insulin proteins leave the golgi and go into the extracellular space via ___ secretion

regulated