Lecture 36: Protein Targeting Flashcards
protein targeting (just in eukaryotic cells)
gest proteins where they need to go
glycosylation
protein synth signal sequences
signal sequences at N terminus
signal sequences at N terminus
of nascent polypeps
direct protein transport
How do signal sequences at N terminus direct protein transport?
by interacting with receptors and processing enzymes
signal sequences are 10-30 aas long
highly conserved
Signal Recognition Particle (SRP)
directs ribosome w/ nascent polypets destinred for:
secretion
insertiion into plasma membrane
or inclusion into lysosomes
to ER to complete protein synth
what do the 3 vesicle transport systems do?
sort ER localized proteins (many are glycosylated) to various destinations by packing into vesciles
what are the 3 vesicle transport systems
COPI, COPII, clathrin
ubiquitinated proteins…
are recycled by proteasomes in order to regulate protein turn over
Function of N-terminal sequence in protein transport
direct protien to right subcellular compartment
how? bind receptor protein
most signal sequences…
15-35 amino acids
10-15 aas are hydrophobic
have either lisine or argenine at beginning (POSTIVELY CHARGED)
what can hydrophobic aas do?
transverse the ER membrane
where is the N terminus
FIRST
Why lisine or argenine?
they are positively charged!
so pos charged residue followed by hydrophobic stuff
What marks the C terminal end of sequence
protease cleaveage site
why are signal sequences cleaved and what by
cleaved by specific proteases
in order to generate a new N-terminus in mature polypeptide
signal sequence
positive charge (lys or arg) followed by hydrophobic amino acids
targets protein with this sequence to the ER
if no signal sequence
protein is retained in cytoplasm
So proteins destined for ____1_____ are insterted into ___2__ by ___3___
1: secretion, integration into plasma membrane, inclusion into lysosomes
2: lumen of ER
3: SRP (signal recognition complex)
signal sequence directs protein transport
signal peptide of end terminus synthed first (N terminus)
hydrophobic sequence recognized, tells ribsome to bind to ER membrane
where is rest of growing protein inserted?
through membrane protein
so that growing polypep chain can keep growing in ER
if you take a cytosolic protein and manipulate it to encode an end terminal signal peptide on the protein…
protein will go into the ER!!!
if you take a protein that usually has ER signal sequence (should be in ER) and remove the signal sequence…
it stays in the cytosol
Based on these experimental data, are signal sequences necessary BUT NOT sufficient (to direct localization) or necessary AND sufficient?
necessary AND sufficient
if it has tag it goes into ER, if it doesn’t it doesn’t, stays in cytoplasm
this tag is enough, doesn’t need other properties
SPR cycle does what
inserts proteins into ER lumen
if a signal sequence is present on growing polypep chain at n terminus…
SRP recognizes it and it interacts with the SRP receptor on ER membrane
as polypep grows, it goes through pore and ends up in ER
this is driven by GTP
how long is protein synth halted
till ribosome, mRNA, and SRP-polypep complex is bound to peptide translocation complex
What biochemical property of signal sequences facilitattes binding to the translocon protein?
hydrophobic property!!!!
SRP cycle slide 6
slide 6
what is synthed frist
signal sequence
recognized by SRP
interaction with translocation complex in ER membrane
docking of ribosome happens
newly synthed chain ends up in lumen
do mature proteins retain the signal peptide
No, they are processed
how are proteins processed?
by protease (cleaves proteins) mature end terminus and mature protein left in ER
glycosylation
adding of sugar units to proteins
happens in ER
what do we do with glycolyated proteins
transport them into plasma membrane
they are used in membrane for cell recognition function
why glycosylate proteins?
for cell cell recognition
happens AS synthesized
NOT in RNA code!
things that happen in ER
glycosylation
formation of disulfide bonds between SH and cystine residues
how to glycosylated
link sugar unit to aspariagine residue of protein
sugar first binds in cytosol
process completed in ER
core glycan
the first sugars that are added
How are proteins degredaded
proteosomes
lysosomes (not talked about much, low pH, have proteases in them, degrade more than just proteins)
proteosomes
just degrade proteins
recognize proteins for degredation because tagged with ubiquitin
allow cell to regulate proten levels
cellular protein life times
finite
degraded at end
tagged
ubiqutin
small (76 amino acids)
covalently linked to lysine residues in proteins marked for destruction
very well conserved across species
important features of ubiquitin
lysine residues
cc-terminus
importance of lysine residues
sites where ubiqutin can be conjucated to other ubiquitin molecs so chains of ubiquitin molecs can form
What enzymes ubiquinate
E1
E2
E3
target proteins for ubiquitination for degredation
E1
attached ubiquitin to itself
forms covalent bond
E2
ubiquitin transferred here
another covalent bond formed so that Ub is bound to E2 enzymes
E3
E3 binds to E2 and substrate
Substrate is protein tagged for degradation here
making a chain of ubiquitin
after E3 step…
ubiquitin transferred from E2 to substrate FOUR times (at least) to make chain of ubiqutin molecs attached to substrate
what is the substrate
the protein tagged for degredation!
need at least 4 Ubs attached for the protein to be marked for degredation
what happens after substrate is marked by at least 4 Ubs
signal for degredation sent
proteosme protyolizes proteins, degrades it (using ATP)
Ubs recycled so it can happen again
Proteosome structure
20S core
two 19 S caps (one per end)
what to the caps on the proteosome do?
can cause conformation change and open to let the protein to be degraded in
they have binding sites for ubiquitinated proteins
encode ATP hydrolyzing enzymes
proteasome core
protease sites are here
proteolyze protein down to peptides (which are released when cap opens up again)
The inner chamber contains the protease enzymes, what doe the outer chamber to do “prepare” proteins for degredation?
1) cap recognizes targeted ubiquitinated proteins
2) unfolds the protein so sites are accessible for proteases in the core
how many kinds of proteases?
Just one!
uses ATP for unfolding
general because all it has to recognize is the ubiqutinated part
Ubiquitinatation requires 3 classes of enzymes
1) E1 enzymes ACTIVATE ubiquitin (C-terminus)
transfer to
2) E2 enzymes CONJUGATE ubiquitin to target proteins at lysine residues
interact with
3) E3 enzymes RECOGNIZE TARGET PROTEINS and facilitate ubiquitination by forming complexes with E2 enzymes
remember… why do we need lysine and C terminus
to form the ubiquitin chain
so how do we ID how long the half life of a protein is?
based on what the amino acid after Met is (b/c Met is often taken off)
really brief overview of ubiquitination
Ub is added to E1, transferred to E2, interaction between E2 and E3 happens, Ub transferred to target protein
N-end rule
identifies proteins with N-terminal amino acid after Met is removed
tells us how long the protein will be around in cells
Explain why humans have 2 E1 genes, 40 E2 genes, but 400 E3 genes.
E1 works independently of the target protein, just works with Ub
E2 enzymes have to interact with LOTS of E3 genes
E3: lots of genes so that you can degrade a lot.
