Lecture 9: Protein sorting Flashcards
what are the three mechanisms that proteins are sorted by & where do most proteins remain
1) transport through nuclear pores
2) transport across membranes (ER, mitochondria, chloroplast)
3) transport by vesicles (lec 10)
most proteins remain in the cytosol
signal sequences purpose
necessary and sufficient to direct a protein to its destination
architecture of the nuclear envelope
what types of molecules have to get in/out of the nucleus?
in: mRNAs, tRNAs, ribosome subunits
out: many proteins that interact with the genome, like DNA polymerase, RNA polymerase, transcription factors
what types of proteins can pass through nuclear pores?
very small proteins (about 5 kD) can passively diffuse through nuclear pores, but most proteins do not
many bigger proteins do enter the nucleus through these pores, but they require an active process of shuttling
process of nuclear transport
- prospective nuclear protein (cargo) is attached to the nuclear localization signal (NLS)
- nuclear import receptor binds to NLS + cargo
- together, the protein + nuclear import receptor move through the nuclear pore
steps of Ran-GTP hydrolysis driving nuclear transport
- Ran GTP is hydrolyzed and Ran-GDP dissociates from receptor
- protein binds to receptor + enter the nucleus from the cytosol + gets released from receptor
- Ran-GTP binds to receptor and exits through the nuclear pore
high Ran-GTP required in the NUCLEUS; GTP hydrolysis in the CYTOSOL
How is Ran GTP hydrolysis and GDP/GTP exchange controlled by other proteins (GAP & GEF)
Ran-GAP -> associated with cytosolic fibrils of nuclear pore; outside of nucleus -> attaches Ran-GDP
Ran-GEF -> associated with nuclear chromatin (DNA); inside of nucleus -> exchanges Ran-GDP and attaches Ran-GTP
what would happen if you depleted the cell of Ran GAP
GTP would not hydrolyze; Ran would accumulate in the cytosol bound to the nuclear transport receptor
ask ab in tutoring
transport into the mitochondria
- protein is synthesized and folds in cytosol (w/ signal sequence)
- protein + SS binds to import receptor protein + attached protein translocator in outer membrane
- protein unfolds as it translocates and moves along the membrane until it reaches the protein translocator in the inner membrane
- protein is tranlocated across both membranes simultaneously, then it refolds inside the mitochondria
- signal peptide is cleaved from mature mitochondrial protein
transport into the ER (aka “co-translational translocation”)
SRP = signal recognition particle
- SRP binds signal sequence and slows/pauses translation
- SRP binds SRP receptor on ER
- SRP leaves and ribosome engages translocation channel
- translation resumes translocating protein across bilayer
what side of ER membrane are the N-terminal signal sequences are cleaved (removed)?
lumenal side of ER membrane
SS is cleaved off by “signal peptidase”
if there is more than one signal sequence in a protein…
the first is called a “start-transfer” sequence and the second signal sequence is called a “stop-transfer” sequence
this mechanism defines the orientation of the protein in the membrane: N terminus in the lumen
start-transfer sequence gets cleaved
do internal signal sequences get cleaved by signal peptidase?
no; placement of SS defines a different orientation in the membrane
multipass membrane proteins contain multiple stop-transfer/start-transfer sequences
protein glycosylation
called “N-linked” glycosylation because the sugar chain is attached to Nitrogen on the asparagine side-chain; only occurs in the ER lumen
-helps protein folding + solubility
-protects protein in harsh environments
-in some cases facilitates protein sorting
-can participate in biological function of protein
required sequence in protein: Asn-X-Ser/Thr… (x is any amino acid except proline)
unfolded protein response (UPR)
translocated proteins must fold once in the ER, cells sense unfolded proteins through the UPR
