Protein Sorting Flashcards
endosymbiosis
- loss of rigid cell wall in ancient anaerobic archaeon facilitates horizontal gene transfers
- digestion of other prokaryotes (archaeal and bacterial) further increases horizontal gene transfers and cell membrane folds in forming primitive nuclear membrane
- aerobic bacterium is engulfed (retains both membranes and its DNA) = mitochondrion
- chloroplasts are created when a photosynthetic bacterium later is engulfed
endosymbiosis
- where did the inner/outer membranes originate
- what is the matrix equivalent to an intact cell
- what do these previous answers mean for carbohydrate metabolism in bacteria
- why do mitochondria contain DNA
- what genes are in the mitochondrial genome
- why is the universal code not used
- why is this not equivalent to any other compartment
- inner- original bacteria; outer- cell that engulfed (primitive eukaryote)
- cytosol
- took place in plasma membrane (outer) in bacteria
- they were original intact cells
- just the important ones necessary to perform functions in the mitochondria; not enough to sustain life
- mitochondria was engulfed before current genetic code was the universal code
- it evolved parallel to the nucleus; mitochondria were world of their own -what happens in one compartment doesn’t have to happen in the other. mutation rates in mitochondria DNA is much greater than nuclear genome
Gated transport:
- facilitator
- compartments bridged
- compartment topology
- membrane crossed
- nuclear pore complex (NPC)
- nucleus/cytosol
- equivalent
- Yes
Transmembrane
- facilitator
- compartments bridged
- compartment topology
- membrane crossed
- translocator proteins
- cytosol –> organelle
- different
- Yes
Vesicular
- facilitator
- compartments bridged
- compartment topology
- membrane crossed
- vesicle
- organelle –> organelle
- equivalent
- No
sorting signals
- located on cargo to be moved direct traffic flow
- signal patch consolidates many scattered sequences (next to each other after protein folding)
- biochemical properties are more important than amino acid identity
- recognized by complementary receptors on targeted organelle
- necessary and sufficient for protein targeting
- may be removed by a signal peptidase after protein reaches final destination
nuclear import signal sequence
rich in K and R
+ charge
located internally
nuclear export signal sequence
amphipathic
located internally
mitochondria import signal sequence
+ charge
located on N-terminus
ER import signal sequence
hydrophobic core
located on N-terminus
ER return signal sequence
KDEL
helps proteins stay in ER or helps to return to ER if escapes
mixed biochemical features
located on C-terminus
nucleus to cytosol uses what signal
nuclear export signal (NES)
cytosol to nucleus uses what signal
nuclear localization signal (NLS)
Nuclear pore complex (NPC) are made up of
nucleoporins (NUPs)
types of nucleoporins (NUPs)
- scaffold: structural, stabilize membrane
- ring: anchor to membrane
- channel: connectors and gates (FG repeats)
- fibrils: neither structural or symmetrical; interact with proteins
nuclear basket
tethers active genes to the NPC
interact with nuclear lamins
regulation of transport
nuclear import
- sorting signal
- amino acids
- location
- recognized by what
- nuclear localization signal (NLS)
- charged amino acids (K, R)
- multiple internal sites
- Nuclear Import Receptors (NIR): cytosolic proteins; ferry cargo into nucleus; also called importins; direct or indirect recognition
nuclear export
- sorting signal
- biochemical property
- location
- recognized by what
- nuclear export signal (NES)
- amphipathic helices
- internal
- nuclear export receptors (NER): nuclear proteins; ferry cargo into cytosol; recycled back to nucleus; also called exportins; recognition must be indirect when cargo is RNA
shuttling
nucleus to cytosol and back
these cargo proteins may have both and NES and NLS
regulation of recognized signal sequence can be by
- phosphorylation
- proteolysis
- inhibitor proteins
importins and exportins are collectively called
karyopherins
when engery is need for nucleus cytosol movement, what type of energy is used
GTP
Ran
monomeric G protein (GTPase)
Ran-GAP
found in cytoplasm
catalyzes removal of phosphate bound to Ran-GTP
Ran-GEF
found in nucleus
exchange our spent GDP for GTP
Ran-GTP
Ran-GDP
found in nucleus
found in cytosol
Nuclear export role of Ran-GTP
- association of Ran-GTP with the exportin (NES) increases affinity for cargo –> cargo binds
- hydrolysis of GTP in cytoplasm causes release of the cargo
nuclear import role of Ran-GTP
- protein with NLS (importin) binds with nuclear import receptor
- once imported, association with Ran-GTP releases the cargo in the nucleus
- Ran-GTP is used to ship importin back into cytosol
- when the importin is recycled to cytosol, it releases Ran-GDP
mitochondrial genome is not complete, most mitochondrial proteins are made from
nuclear genes
TOM
- Translocase of the Outer Mitochondrial membrane
- beta barrel
- regulated by cytosolic kinases
- cytosol –> intermembrane space
- cytosol –> outer membrane-
TIM
- Translocase of the Inner Mitochondrial membrane
- intermembrane space –> matrix
- intermembrane space –> inner membrane
cytosolic chaperone proteins
keep mitochondrial-destined polypeptides unfolded
e.g. Hsp70/Hsp40
SAM
- Sorting and Assembly Machinery of outer mitochondrial membrane
- beta barrel pore
- receives proteins from TOM and Small TIM and inserts beta-barrel proteins into OM
MIM
- Mitochondrial Import complex
- receives proteins from TOM and inserts alpha-helical transmembrane proteins into outer membrane
beta-signal
- found on first beta strand inserted in SAM complex
- not cleaved after insertion into OM
signal-anchor domain
- sorting signal for alpha-helical transmembrane proteins
- hydrophobic amino acids with + charge at the N-end
- not cleaved after insertion into the OM
- N-terminal use MIM
- Internal use SAM
- single-pass uses MIM or SAM
- multi-pass uses MIM/TOM70
what kind of proteins are Small Tim proteins in the Intermembrane Space
chaperone
shuttle proteins to SAM and TIM 22
MIA
- Mitochondrial Import and Assembly complex
- redox reactions
- cysteine rich sorting sequence
TIM23
- Translocase of the Inner Mitochondrial membrane
- workhorse: primary carrier into inner membrane
- inserts most IM
TIM 22
- Translocase of the Inner Mitochondrial membrane
- inserts some metabolite carrier proteins involved in carbohydrate chemistry
- inserts TIM 23
How do proteins get to TIM22
- through TOM and small TIM
- sorting sequence is not removed
how do proteins get to TIM23
- directly from TOM
- sorting sequence is removed after insertion into IM
OXA
Oxidase Assembly translocase complex
- export protein: if protein goes all the way into matrix from TIM23, OXA can export out of matrix and into IM
- OXA can also export proteins that were made in the mitochondria
how do proteins gets all the way into the matrix of mitochondria
TOM –> TIM23 –> PAM
requires ATP
PAM
- Presequence translocase-Associated Motor complex
- recognizes sorting sequence: amphipathic helix that is cleaved after import
in transit from cytosol to matrix, what processes requires ATP
- dissociation of cytosolic Hsp70
- transport through TIM23 relies on membrane gradient
- release step of mitochondrial Hsp70 “pulling” into matrix
- mitochondrial Hsp60 helps protein fold once its in the matrix
cytosol –> ER: two methods of insertion
co-translational
post-translational
co-translational insertion into ER
- happens on rough ER
- requires: Signal Recognition Particle (SRP), SRP receptor, and Sec61 gated translocator
post-translational insertion into ER
- happens when translation occurs on free ribosomes
- requires: cytosolic chaperone proteins; Sec61 gated translocator, ER lumen chaperone protein (Binding Protein -BiP
Co-translational insertion into ER
- cytosolic chaperone
- energy source
- energy use
- SRP/SRP receptor
- GTP
- SRP recycling, translation elongation
post-translational insertion into ER
- cytosolic chaperone
- energy source
- energy use
- Hsp70/Hsp40
- ATP
- ER lumen chaperone protein (Binding Protein-BiP (pulls in)
sorting sequence for cytosol –> ER
- located at N-terminus
- 3 parts: N-domain is charge, H-DOMAIN IS HYDROPHOBIC, and C-domain is polar
- cleaved in ER lumen by signal peptidase
Signal Recognition Particle
- contains 1 noncoding RNA and 6 proteins
- signal sequence binding pocket is hydrophobic because sorting sequence is hydrophobic also
steps of cytosol –> ER
- SRP binding to signal sequence pauses translation (blocks A and exit sites)
- bind to SRP receptor on surface of ER
- SRP releases and allows signal sequence to enter into translocator and ribosome stays with translocator
- SRP and SRP receptor are releases and recycled
- SRP and SRP receptor are GTPases but don’t use GEF/GAPs
ER transmembrane proteins have two signal sequences
- N-terminal start signal (hydrophobic)
- stop-transfer sequence (hydrophobic)
these two sequences together span the membrane
are start-signal sequences or stop-signal sequences more hydrophobic
start-signal
proteins and lipids are glycosylated in the
ER
- sugar chains signal a mature protein and allows proteins to be folded and misfolded proteins to be recognized
- sugar always face away from cytosol (in ER…face lumen, on cell membrane…face outside)
