Exam 3: Ch 13 Targeting & Insertion of Proteins (ER) Flashcards Preview

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Flashcards in Exam 3: Ch 13 Targeting & Insertion of Proteins (ER) Deck (65):
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protein targeting

delivery of newly synthesized proteins to their proper destination

2 kinds of process

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2 processes of protein targeting

signal-based targeting

vesible-based trafficking (secretory pathway)

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signal-based targeting

protein from cytoplasm --> intracellular organelle

occurs during translation or soon after

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for memb. proteins, signal-based targeting leads to...

insertion of the protein into the lipid bilayer

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for water-soluble proteins, signal-based targeting leads to...

translocation of the protein across the membrane into the aqueous interior of an organelle

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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

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synthesis of all proteins begins by ______ ribosomes

cytosolic

if no signal peptide then product released into cytoplasm

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memb. bound proteins and some organelles have a _____ peptide at the _______ terminus

signal peptide, amino terminus

ex. lysosome/secreted proteins

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is a signal peptide usually cleaved off later?

yes

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SRP

signal recognition particle

cytosloic RNP

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is insulin an example of a secreted protein?

yes, has a SRP --> rough ER

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vesicles on Golgi go from the ___ to ____ face and are ____

cis, trans

modified

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targeting to the nucleus occurs how

mature protein contains a nuclear localization signal

pattern of a few aa directs to nucleus

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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

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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

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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)

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each organelle has specific ______ receptors for specific signal sequences

receptors

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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

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ER in general

large organelle made of tubules and flattened sacs

membrane continuous with nuclear membrane

site of lipid and membrane protein biosynthesis

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pulse-chase experiments with purified ER membrane demonstrated...

that secreted proteins cross the ER memb.

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what do you find out with pulse-chase

where a tagged molecule ends up

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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

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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)

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why is it hard to study memb. proteins?

if you break the membrane, the proteins stop working

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a homogenizer uses what to break up rough ER

mild detergent

then a magnet separates microsomes from microsomes with w/ ribosomes

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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

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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

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signal sequences contain 1+ ______ aas adjacent to a stretch of hydrophobic residues

positive

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cotranslational translocation

transport of most secretory proteins into the ER lumen begins while the protein is still bound to the ribosome

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cotranslational translocation is initiated by two GTP hydrolyzing proteins

the signal recognition particles and its receptor located in the ER membrane

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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

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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

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what provides energy of translocation of the proteins into the lumen

energy from chain elongation in the ribosome

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why don't other molecules go through the translocon pore?

there is a short helical plug

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signal peptidase

enzyme in the translocon that cleaves the signal peptide as it enters the lumen of the rough ER

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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

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the final orientation of integral membrane proteins is established during their _______

biosynthesis

during transport, their orientation is preserved

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topogenic sequence

direct membrane insertion and orientation of integral proteins

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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

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single pass protein

only span the membrane once

classes I, II, III, and tail-anchored proteins

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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

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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

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type III protein

same orientation as type I

no signal sequence, anchor sequence near N terminus embeds in rough ER

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multiple pass proteins

class IV - have 2+ membrane spanning segments

g-protein coupled receptors (7 pass protein)

have multiple internal topogenic sequences

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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

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example of type IV protein with N-terminus in cytosol

GLUT transporters (glucose)

ion-channel proteins

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example of type IV protein with N terminus that is extracellular

G protein coupled receptors (7 transmemb. domains)

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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

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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

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peripheral membane protein

don't cross membrane

may be synthesized by rough ER or free ribosomes

sticks to memb. by phospholipid

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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

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glycoprotein

a protein with an attached carbohydrate - begin in rough ER, modified in golgi

glycosylation occurs on extracellular domain

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o-linked oligosaccharide

glycoprotein with carbohydrate chain attached to OH group

serine/threonine

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n-linked oligosaccharide

glycoprotein with carbohydrate chain attached to amide nitrogen

asparagine

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oligosaccharide side chains may promote the _______ and ________ of glycoproteins

folding, stability

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the lumen is an ______ environment, while the cytoplasm is a _______ environment

oxidized, reduced

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disulfide bonds make a protein more ______

stable

happens in an oxidation rxn (only in lumen of rough ER)

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disulfide bonds are only found in which types of proteins

soluble secretory proteins

extracellular domains of memb. proteins (ex. insulin)

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which aa forms disulfide bonds

cysteines

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what do lectins in the rough ER lumen do

help fold proteins with n-linked oligosaccharides

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assembly of multisubunit proteins occurs in the ________ __

rough ER

ex. immunoglobulins

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dislocation

when misfolded proteins in the ER are targeted for transport to cytosol for degradation

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ERAD

ER associated degradation complex

channel for dislocation of misfolded proteins

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p97

member of the AAA ATPase family that couples ATP hydrolysis to disassemble proteins

targets ubiquitin on misfolded proteins

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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