Lecture 9 - quiz 1

Question 1

how can cellular membranes be made

Answer 1

by expanding pre existing membranes not de novo

Question 1

describe how all proteins encoded

Answer 1

by nuclear genes and translated in cytosol
few exceptions (mito)

Question 2

when must proteins be sorted

Answer 2

during or after translation to their correct compartment or membrane
sorted by signals

Question 3

where is sorting info stored

Answer 3

inside proteins themselves
in sequence of proteins - determines where, ptms and folding

Question 4

how are proteins transported across pore

Answer 4

if folded = hard to be transported across pore
usually co translationally = before has folded
many ribosomes attached to er membrane
if folded = hard to move through

Question 5

describe what happens when secretory pathway proteins are inserted into or across er membrane

Answer 5

transport to further compartments - golgi, pm, enodosmes, lysosomes
outer and inner nuclear membranes are continuous with er

Question 6

describe rough er

Answer 6

many attached ribosomes
secretory protein synthesis

Question 7

describe smooth er

Answer 7

no ribosomes
sites of lipid synthesis

Question 8

describe targeting signals

Answer 8

sequences within a protein that specify its organelle location - zip code or signal peptide - so cell reads and is like you go here
often independent from structure or biochemical function of proteins
recognized by their pattern but not usually an exact sequence, independent of region with hydrophobic patches

Question 9

when are targeting signals removed

Answer 9

may be removed by proteolysis after targeting complete or form part of native structure

Question 10

describe targeting steps - 3

Answer 10

1. recognize a signal on a protein
2. connect protein to the membrane
3. translocate protein in to or across the membrane

Question 11

describe signal hypothesis

Answer 11

secretory proteins were known to enter er during translation of their mrnas

Question 12

describe signal hypothesis observation

Answer 12

a newly translated secretory protein is larger than its final form

Question 13

describe signal hypothesis hypothesis

Answer 13

extra sequence is targeting signal peptide whose main function is to direct insertion into er
signal peptide must start mechanism to connect ribosome to translocation pore
signal peptide is cleaved off after targeting is finished

Question 14

describe describe signal hypothesis experimental approach

Answer 14

if has ribosomes - rna encoding for secretory pathway = then dissociated
if has er too = now nascent peptide recognized by pore = pushed into er

Question 15

describe ribosome exit tunnel

Answer 15

Nascent polypeptides exit the ribosome through a tunnel in the large (60s)
subunit
Tunnel is neutral, polar, too small for tertiary folding
Surface around exit site provides binding sites for ER targeting mechanisms
30 to 40 amino acids of nascent polypeptide between peptidyl- transferase site and the exit

Question 16

describe how secretory signal peptides direct proteins

Answer 16

to er for translocation into or across membrane
co translationally mostly

Question 17

what do many secretory pathway proteins have

Answer 17

additional targeting sequences that direct them to organelles
often a polypeptide sequence pattern - motif
sometimes a ptm

Question 18

describe organelles not in secretory pathway

Answer 18

have their own targeting signals

Question 19

describe signal peptide pattern

Answer 19

hydrophobic central region 8 or more residues long with short polar regions on each side

Question 20

where are signal peptides in most cases

Answer 20

at N terminus = do not want protein to fold in cytosol
shorter hydrophobic regions - 8-16 residues
often cleaves off after translocation

Question 21

describe signal anchors

Answer 21

signal peptides that also become tm helices
not cleaved off
can be in diff places in protein
longer hydrophobic region = 18-24 residues

Question 22

describe signal peptide –> targeting steps - step 1

Answer 22

1. recognize a signal on a newly translated protein= ribosome begins translating polypeptide with a signal, Signal Recognition Particle (SRP) is a soluble protein that binds signal and ribosome during translation

Question 23

describe signal peptide –> targeting steps - step 2

Answer 23

2. connect protein to the membrane= SRP Receptor (SRP-R) is a membrane protein that binds the ribosome-SRP complex, SRP-R links ribosome to translocon pore in ER

Question 24

describe signal peptide –> targeting steps - step 3

Answer 24

3. translocate protein into or across the membrane= energy of translation on ribosome drives polypeptide through the translocon

Question 25

describe signal peptide –> targeting steps - generally

Answer 25

signal peptide part of primary sequence but not native state of protein
if no signal = stays in cytosol and becomes cytosol protein

Question 26

what is srp

Answer 26

Ribonucleoprotein: 6 protein subunits and 1 RNA
Signal sequence recognition subunit with GTPase activity
Translation regulatory domain at the opposite end
RNA strand forms flexible linker

Question 27

describe ribosome to srp - 2 steps

Answer 27

1 - srp samples all nascent polypeptides that emerge from ribosomes
2 - when signal peptide recognized = srp attaches tightly to both signal and ribosome
- srp pauses translation at ribosome and binds gtp
hydrophobic characteristics of targeting signal= bind to them

Question 28

describe srp-r to translocon - 3 steps

Answer 28

3 - The ribosome-SRP complex binds to the SRP-R on ER
4 - Ribosome moves to the translocon and becomes tightly bound
5 - SRP and SRP-R dissociate from ribosome = translation resumes, and polypeptide translocates into lumen – lumenal polypeptide does not contact the cytosol

Question 29

describe srp and srp-r - steps

Answer 29

Step 2: SRP attached to ribosomes is in the GTP-bound state
* Step 3: SRP-R is also a GTPase, and is in the GTP-bound state when it
recognizes SRP-ribosomes
* Step 5: GTP hydrolysis by both SRP and SRP-R dissociate them and
recycle them
* GTP is used like a “switch”

Question 30

describe er translocon

Answer 30

sec61 complex
protein has 2 parts - yellow and blue
form both sides of aq pore
the 2 parts of pore open laterally to integrate tm helices into membrane

Question 31

describe pore - er translocon

Answer 31

inactive pore = plugged by part of protein
needs signal peptide to push away plug
active pore is open but tightly sealed on ribosome
inside of pore = neutral, polar

Question 32

describe translocation of lumenal protein - step 1

Answer 32

Signal peptide triggers opening of the translocon

Question 33

describe translocation of lumenal protein - step 2

Answer 33

Polypeptides are translocated in an extended, unfolded state
Movement of polypeptide is driven by energy of translation pushing it out of the ribosome

Question 34

describe translocation of lumenal protein - step 3

Answer 34

Signal peptidase often removes signal peptide during translocation – not sequence-specific but has a preferred site

Question 35

describe integration of tm helix - gen

Answer 35

protein with n terminal signal sequence and tm helix
hydrophobic start transfer peptide binding site
stops process of translocation
protein anchored in membrane

Question 36

describe integration of tm helix - steps 1-3

Answer 36

1 - signal peptide starts translocation of lumenal part
2 - tm helix is recognized by translocon and integrated laterally into membrane during translation
3 - cytosolic part is translated in cytosol

Question 37

describe type 1 tm proteins

Answer 37

N in lumen
C in cytosol
most are like this but not all since sometimes want n in cytosol

Question 38

describe type 2 tm proteins

Answer 38

n terminal in cytosol and c in lumen
direction translocation happening in
as being synthesized = pushed inside lumen

Question 39

what is signal anchor

Answer 39

ignal peptide with long hydrophobic region that is not cleaved off, but becomes the TM domain- length of the hydrophobic region is 18-24 Amino Acids

Question 40

describe signal anchor integration - steps 1-3

Answer 40

1 - Signal anchor opens translocon like a signal peptide
2 - translocon recognizes charges next to the signal anchor to determine orientation in membrane - positive charges (from aas that surround hydrophobic patch) in cytosol, neg in lumen
3 - signal anchor is recognized as tm domain and integrated laterally - inserted into membrane

Question 41

describe multipass tm proteins

Answer 41

combos of signal anchor and tm helices cause alternating orientation of multipass tm proteins

Question 42

describe topology of multipass tm proteins

Answer 42

tm organization of secretory pathway proteins can often be predicted =
hydrophobicity - number of tm helices
charge distribution - orientation in membrane
can predict other modifications - disulfide bonds, glycosylation, ubiquitination

Question 43

describe N linked glycosylation motif

Answer 43

on asn - asparagine - side chain in context of Asn-X-Ser-Thr motif
the same glycan is always attached at er
mostly mannose with 3 glucoses
motif recognized for it to be glycosylated

Question 44

describe N

Answer 44

n linked glycosylation on asn side chain amide - gln not recognized by OST

Question 45

describe N-x-S/T motif modifications

Answer 45

most N-x-S/T motifs in lumen are modified depending on accessibility to ost
glycans can be modified after addition but are not removed until protein degraded
(permanent modification)

Question 46

describe glycosylation process - steps 1-4

Answer 46

1 - Oligosaccharides are synthesized attached to a specialized lipid in the ER
2 - OST attaches glycan during translocation (tm protein attaches to membrane of er, recognizes in specific context)
3 - State of glycan is used as a signal in ER quality control of folding (enzyme will take glycogen and add glycogen, same for all proteins = signal that protein belongs to secretory pathway)
4 - Glycan is modified in Golgi after exit from ER

Question 47

describe N linked glycosylation

Answer 47

most secretory proteins have oligosaccharides - glycans - covalently attached =
helps stabilize the native state
protect against proteases
function in cell surface signalling
what happens to proteins inserted into er

Question 48

what is OST

Answer 48

oligosaccahryl transferase