Lecture 9 - quiz 1 Flashcards
how can cellular membranes be made
by expanding pre existing membranes not de novo
describe how all proteins encoded
by nuclear genes and translated in cytosol
few exceptions (mito)
when must proteins be sorted
during or after translation to their correct compartment or membrane
sorted by signals
where is sorting info stored
inside proteins themselves
in sequence of proteins - determines where, ptms and folding
how are proteins transported across pore
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
describe what happens when secretory pathway proteins are inserted into or across er membrane
transport to further compartments - golgi, pm, enodosmes, lysosomes
outer and inner nuclear membranes are continuous with er
describe rough er
many attached ribosomes
secretory protein synthesis
describe smooth er
no ribosomes
sites of lipid synthesis
describe targeting signals
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
when are targeting signals removed
may be removed by proteolysis after targeting complete or form part of native structure
describe targeting steps - 3
- recognize a signal on a protein
- connect protein to the membrane
- translocate protein in to or across the membrane
describe signal hypothesis
secretory proteins were known to enter er during translation of their mrnas
describe signal hypothesis observation
a newly translated secretory protein is larger than its final form
describe signal hypothesis hypothesis
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
describe describe signal hypothesis experimental approach
if has ribosomes - rna encoding for secretory pathway = then dissociated
if has er too = now nascent peptide recognized by pore = pushed into er
describe ribosome exit tunnel
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
describe how secretory signal peptides direct proteins
to er for translocation into or across membrane
co translationally mostly
what do many secretory pathway proteins have
additional targeting sequences that direct them to organelles
often a polypeptide sequence pattern - motif
sometimes a ptm
describe organelles not in secretory pathway
have their own targeting signals
describe signal peptide pattern
hydrophobic central region 8 or more residues long with short polar regions on each side
where are signal peptides in most cases
at N terminus = do not want protein to fold in cytosol
shorter hydrophobic regions - 8-16 residues
often cleaves off after translocation
describe signal anchors
signal peptides that also become tm helices
not cleaved off
can be in diff places in protein
longer hydrophobic region = 18-24 residues
describe signal peptide –> targeting steps - step 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
describe signal peptide –> targeting steps - step 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
describe signal peptide –> targeting steps - step 3
- translocate protein into or across the membrane= energy of translation on ribosome drives polypeptide through the translocon
describe signal peptide –> targeting steps - generally
signal peptide part of primary sequence but not native state of protein
if no signal = stays in cytosol and becomes cytosol protein
what is srp
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
describe ribosome to srp - 2 steps
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
describe srp-r to translocon - 3 steps
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
describe srp and srp-r - steps
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”
describe er translocon
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
describe pore - er translocon
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
describe translocation of lumenal protein - step 1
Signal peptide triggers opening of the translocon
describe translocation of lumenal protein - step 2
Polypeptides are translocated in an extended, unfolded state
Movement of polypeptide is driven by energy of translation pushing it out of the ribosome
describe translocation of lumenal protein - step 3
Signal peptidase often removes signal peptide during translocation – not sequence-specific but has a preferred site
describe integration of tm helix - gen
protein with n terminal signal sequence and tm helix
hydrophobic start transfer peptide binding site
stops process of translocation
protein anchored in membrane
describe integration of tm helix - steps 1-3
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
describe type 1 tm proteins
N in lumen
C in cytosol
most are like this but not all since sometimes want n in cytosol
describe type 2 tm proteins
n terminal in cytosol and c in lumen
direction translocation happening in
as being synthesized = pushed inside lumen
what is signal anchor
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
describe signal anchor integration - steps 1-3
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
describe multipass tm proteins
combos of signal anchor and tm helices cause alternating orientation of multipass tm proteins
describe topology of multipass tm proteins
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
describe N linked glycosylation motif
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
describe N
n linked glycosylation on asn side chain amide - gln not recognized by OST
describe N-x-S/T motif modifications
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)
describe glycosylation process - steps 1-4
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
describe N linked glycosylation
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
what is OST
oligosaccahryl transferase
