Exam 3: Ch 13 Targeting & Insertion of Proteins (ER) Flashcards
protein targeting
delivery of newly synthesized proteins to their proper destination
2 kinds of process
2 processes of protein targeting
signal-based targeting
vesible-based trafficking (secretory pathway)
signal-based targeting
protein from cytoplasm –> intracellular organelle
occurs during translation or soon after
for memb. proteins, signal-based targeting leads to…
insertion of the protein into the lipid bilayer
for water-soluble proteins, signal-based targeting leads to…
translocation of the protein across the membrane into the aqueous interior of an organelle
secretory pathway (vesible-based trafficking)
transport of proteins from the ER using vesicles
destination may be outside the cell
integral memb. proteins transported to golgi, lysosome, and membrane
synthesis of all proteins begins by ______ ribosomes
cytosolic
if no signal peptide then product released into cytoplasm
memb. bound proteins and some organelles have a _____ peptide at the _______ terminus
signal peptide, amino terminus
ex. lysosome/secreted proteins
is a signal peptide usually cleaved off later?
yes
SRP
signal recognition particle
cytosloic RNP
is insulin an example of a secreted protein?
yes, has a SRP –> rough ER
vesicles on Golgi go from the ___ to ____ face and are ____
cis, trans
modified
targeting to the nucleus occurs how
mature protein contains a nuclear localization signal
pattern of a few aa directs to nucleus
how to test nuclear localization
put nuclear localization signal into a diff protein and see if it ends up in the nucleus
some TF are held inactive in the cytoplasm until another protein finds that has a nuclear localization signal
targeting to the ER in general
involves nascent proteins being synthesized on a ribosome –> when finished, extruded into ER memb. –> lumen
in lumen, chaperones help fold proteins
folded proteins undergo post-translational modifications –> transported out of ER
signal sequence (uptake-targeting sequence/signal peptide)
~20aa in the sequence of the protein itself
directs targeting to a particular organelle destination
found at N terminus (1st part of protein synthesized)
each organelle has specific ______ receptors for specific signal sequences
receptors
translocation channel
allows the targeted protein to enter the target organelle through the membrane
occurs after the signal sequence has interacted w/ the receptor on the organelle
ER in general
large organelle made of tubules and flattened sacs
membrane continuous with nuclear membrane
site of lipid and membrane protein biosynthesis
pulse-chase experiments with purified ER membrane demonstrated…
that secreted proteins cross the ER memb.
what do you find out with pulse-chase
where a tagged molecule ends up
pulse-chase experiment with radiolabeled aa
pulse: add radiolabeled aa glycine that gets incorporated into secretory proteins in rough ER
collect samples and homogenize them
chase: add lots of unlabeled glycine
microsome
little portions of rough ER memb. after cell is broken open (homogenized)
capable of protein translocation
showed that proteins synthesized by ribosomes on outside end up on the inside of the microsome (rough ER)
why is it hard to study memb. proteins?
if you break the membrane, the proteins stop working
a homogenizer uses what to break up rough ER
mild detergent
then a magnet separates microsomes from microsomes with w/ ribosomes
pulse-chase w/ insulin
homogenize –> microsomes then ribosome Ab to separate rough ER microsomes from others
grind everything with detergent then insulin Ab to isolate insulin and analyze
shows newly synthesized insulin in rough ER microsomes 1st, then insulin goes somewhere else
after synthesis of a secretory protein begins on free ribosomes in the cytosol, an __ signal sequence in the nascent protein directs the _______ to the ER membrane and initiates ______ of the growing pp across the ER membrane
ER, ribosomes, translocation
signal sequences contain 1+ ______ aas adjacent to a stretch of hydrophobic residues
positive
cotranslational translocation
transport of most secretory proteins into the ER lumen begins while the protein is still bound to the ribosome
cotranslational translocation is initiated by two GTP hydrolyzing proteins
the signal recognition particles and its receptor located in the ER membrane
what does SRP do
binds the ER signal sequence in a growing pp and the large ribosomal subunit
brings this complex to the ER membrane and docks to SRP receptor
translocon
a complex of proteins that forms a channel in the Er membrane
growing pp goes through the central pore of the translocon into ER lumen
what provides energy of translocation of the proteins into the lumen
energy from chain elongation in the ribosome
why don’t other molecules go through the translocon pore?
there is a short helical plug
signal peptidase
enzyme in the translocon that cleaves the signal peptide as it enters the lumen of the rough ER
post-translational translocation
secretory proteins in yeast enter ER lumen after translation has been completed
doesn’t use SRP, only the translocon + signal peptide
uses ATP hydrolysis as the driving force
the final orientation of integral membrane proteins is established during their _______
biosynthesis
during transport, their orientation is preserved
topogenic sequence
direct membrane insertion and orientation of integral proteins
what does the topology of a memb. protein refer to
the number of times it spans the memb.
the orientation of the membrane spanning segments
single pass protein
only span the membrane once
classes I, II, III, and tail-anchored proteins
type I protein
3 domains (cytoplasmic c-terminal, transmembrane, extracellular n-terminal)
signal sequence makes them similar to secreted proteins
have a stop transfer sequence
type II protein
no signal sequence
have an anchor sequence in middle that tells ribosomes to embed protein in rough ER
opposite orientation to type I
type III protein
same orientation as type I
no signal sequence, anchor sequence near N terminus embeds in rough ER
multiple pass proteins
class IV - have 2+ membrane spanning segments
g-protein coupled receptors (7 pass protein)
have multiple internal topogenic sequences
stop transfer anchor sequence
type I proteins
tells translocon to stop transport of mRNA/protein when 22aa hydrophobic sequence is reached
allows the hydrophobic transmembrane segment to be anchored in bilayer
example of type IV protein with N-terminus in cytosol
GLUT transporters (glucose)
ion-channel proteins
example of type IV protein with N terminus that is extracellular
G protein coupled receptors (7 transmemb. domains)
some cell-surface proteins are tethered to the membrane by a ________ anchor
phospholipid anchor called GPI
signal sequence in luminal domain causes this
these proteins can rapidly diffuse laterally
deduce topology of a memb. protein by aa sequence
hydropathic index - hydrophobic aa are positive and hydrophilic aa are negative
ex. HGH is type I
peripheral membane protein
don’t cross membrane
may be synthesized by rough ER or free ribosomes
sticks to memb. by phospholipid
4 modifications membrane and soluble secretory proteins synthesized on the rough ER undergo before reaching their destinations
covalent addition and processing of carbs (glycosylation) in ER and golgi
formation of disulfide bonds in ER
proper folding and assembly in ER
proteolytic cleavages in ER, golgi, and secretory vesicles
glycoprotein
a protein with an attached carbohydrate - begin in rough ER, modified in golgi
glycosylation occurs on extracellular domain
o-linked oligosaccharide
glycoprotein with carbohydrate chain attached to OH group
serine/threonine
n-linked oligosaccharide
glycoprotein with carbohydrate chain attached to amide nitrogen
asparagine
oligosaccharide side chains may promote the _______ and ________ of glycoproteins
folding, stability
the lumen is an ______ environment, while the cytoplasm is a _______ environment
oxidized, reduced
disulfide bonds make a protein more ______
stable
happens in an oxidation rxn (only in lumen of rough ER)
disulfide bonds are only found in which types of proteins
soluble secretory proteins
extracellular domains of memb. proteins (ex. insulin)
which aa forms disulfide bonds
cysteines
what do lectins in the rough ER lumen do
help fold proteins with n-linked oligosaccharides
assembly of multisubunit proteins occurs in the ________ __
rough ER
ex. immunoglobulins
dislocation
when misfolded proteins in the ER are targeted for transport to cytosol for degradation
ERAD
ER associated degradation complex
channel for dislocation of misfolded proteins
p97
member of the AAA ATPase family that couples ATP hydrolysis to disassemble proteins
targets ubiquitin on misfolded proteins
protein cleavage in rough ER
pro-proteins are precursors to the real protein (become proteins after post-translational modifications)
insulin is a pre-pro-protein (pre designates signal sequence)
cleave signal sequence to turn into pro-protein
