exam 3 intracellular protein transport Flashcards
where does translation begin
cytosol
where are proteins synthesized
cytosol, then transported to membrane-enclosed organelles
what does transport rely on
specific signal sequences within the protein primary sequence
what does gated transport do
moves proteins into and out of the nucleus from cytosol through nuclear pore complexes
how are proteins transported into the mitochondira
via transmembrane transport through the TOM and TIM complexes
how are proteins imported into the ER
via co-translational transport
what are eukaryotic cells subdivided into
functionally distinct, membrane-enclosed compartments
what is the cytoplasm
cytosol + organelles
what occurs/is in the cytosol
where protein synthesis and degradation occurs
where proteasome is
where metabolism occurs
where does transmembrane transport go to
mitochondria, ER, plastids, peroxisomes
what is the movement of proteins between organelles consistent with
topological similarities among the compartments
what are topological similarities and what do they allow for
compartments with similar membrane orientations - allows for same transport
what is the movement of proteins between organelles mediated by
sorting signals and receptors
what are the three fundamental mechanisms of transport of proteins
gated, transmembrane, and vesicular transport
what do nuclear pore complexes function as
selective gates that actively transport specific macromolecules and assemblies, which allows for free diffusion of smaller molecules
what is transmembrane transport
protein traffic between the cytosol and an organelle that is topologically different (i.e. cytosol to ER)
what does transmembrane transport occur through
membrane-bound protein translocators
what is vesicular transport
protein traffic among topologically equivalent organelles
where does vesicular transport occur through
membrane-enclosed transport intermediates called vesicles
what organelles do vesicular transport occur between
ER and golgi
golgi and lysosomes
endosomes and plasma membrane
what restricts the passage of large macromolecules in gated transport
nuclearporins lining the central pore containing unstructured regions
how do nuclearporins act as gates
they don’t have a strong tertiary structure, which allows them to move from one conformation to another - energy dependent
what does moving up a concentration gradient require
active transport
how is nuclear import initiated in gated transport
nuclear localization signals within the cargo are recognized by nuclear import receptors
where are nuclear localization signals present
nuclear proteins
what is the signal for nuclear import made up of
five basic amino acids in a row
what does nuclear transport do
concentrates specific proteins in the nucleus, increasing order in the cell and consuming energy
what is the energy in nuclear transport provided by
hydrolysis of GTP by Ran
what is Ran
a small GTPase
what does GEF do for nuclear transport
activates protein
what does GAF do for nuclear transport
speeds up hydrolysis of GTP to GDP to deactivate protein
where is Ran found
in both the cytosol and nucleus
what is Ran required for
both the nuclear import and export systems
what is import deactivated by (gated transport)
RAN-GTP (releases cargo)
what is export activated by (gated transport)
RAN-GTP (picks up cargo)
what does control of nuclear transport depend on
regulation of nuclear localization and export signals
what are the two states Ran exists in
one with GTP attached and one with GDP attached
what is RAN-GEF
nuclear protein - catalyses the binding of GTP to RAN inside the nucleus
what is RAN-GAP
a cytosolic protein - activates hydrolysis of GTP attached to RAN
what creates a RAN-GTP gradient
hydrolysis of GTP attached to RAN because there is more RAN GTP inside the nucleus than outside
what occurs after RAN-GTP binds to the nuclear import receptors
happens after they diffuse through nuclear pore and into nucleus - causes them to release their cargo proteins which accumulate inside nucleus
what is the effect of RAN-GTP on nuclear export receptors
they bind to their cargo
which RAN associates or dissociates
RAN-GDP dissociates from receptors, RAN-GTP binds to receptors
what does binding to RAN cause
release of the cargo and allows the receptor to bind back to nuclear pore and be shuttled outside the nucleus into cytoplasm
where is RAN-GDP in
cytoplasm
where is RAN-GTP in
nucleus - forces release of cargo
what does hydrolysis of GTP to GDP cause
dissociation of RAN from import and export receptors
how is activity of gene regulatory proteins controlled
by keeping them out of the nuclear compartment until they are needed there
how are T cells activated
by antigen binding
why do calcium levels increase
calcium channel opening - activates the protein phosphatase, calcineurin
what does calcineurin dephosphorylate and what does it cause
NFAT - causes a conformational change which exposes a nuclear import sequence on the protein’s surface
what happens with mitochondrial transport
mitochondrial proteins are first fully synthesized as precursor proteins in the cytosol and then translocated into mitochondria
what do most mitochondrial precursor proteins have
a signal sequence at their N terminus that, when folded, forms an amphipathic alpha helix
what residues does a signal sequence at the N terminus have
charged residues clustered on one side, uncharged residues clustered on the other side
what does the TOM complex does
transports across the outer membrane
what does the TIM complex do
transports across the inner membrane (imbedded in inner mitochondrial membrane)
why is TOM considered a gatekeeper
all proteins that are going to get in the mitochondria have to interact with it
what does TOM help with
helps insert transmembrane proteins into outer mitochondrial membrane
what does TIM get proteins into
intermembrane space of mitochondria, including matrix
what does TIM 23 span
both outer and inner mitochondrial membranes
what does TIM transport
soluble proteins into matrix and membrane proteins into inner mitochondrial membrane
what are newly synthesized mitochondrial proteins in the cytosol surrounded by
protein-folding chaperones that prevent them from aggregating
what do mitochondrial versions of chaperones do
help precursor proteins fold into 3D structures once they enter the mitochondria
why does directional transport require energy
it is not energetically favorable
what does mitochondrial protein import require
ATP
what is cytosolic chaperone disassembly driven by
ATP hydrolysis
where are proteins synthesized for both gated and transmembrane transport
cytosol
where does gated transport move through
nuclear pores using nuclear import and export receptors and RAN GDP and GTP cycle****know these for exam
where is the precursor synthesized for mitochondrial transport
the cytosol and kept in slightly unfolded state to be shuttled through TOM and TIM transporters into mitochondria
what is nuclear import done through
GTP hydrolysis
what is mitochondrial import done through
ATP hydrolysis
what is the protein gradient a source of energy for
transporting across mitochondria membrane
what type of transport are gated and transmembrane transport
active
what do proteins entering the ER undergo
Co-translational translocation
how is transport into ER different than transport into nucleus and mitochondria
proteins are not fully synthesized before they are transported into ER
what is happening to proteins simultaneously as they’re going into the ER
they’re being transported as they’re being translated
how do proteins enter the ER
as ribosome is translating the protein, near N-terminus a signal sequence marks this protein as one to be transported into ER
signal pauses translation and directs whole complex to move to ER; when translation restarts, the protein is pushed across the membrane as it’s being translated
what types of proteins require co-translational translocation
water soluble (non membranous) proteins destined to:
- localize to the lumen of any non-nuclear organelle (ER, golgi, lysosomes, etc)
- be secreted out of the cell (e.g. hormones)
what are SRPs
proteins that contain both RNA and polypeptide components
what are the 2 functional domains of SRPs
- translational pause domain, which interacts with ribosome and prevents binding of elongation factors (EF2, etc - which bring in new tRNAs), therefore pausing translation
- signal-sequence-recognizing component
what does signal sequence recognizing component do
stops translation and can be recognized by SRP receptor on ER
what happens if the signal sequence is near the N-terminus as translation happens
very hydrophobic region of the protein that is now accessible and recognized by SRP
what happens when SRP binds to the signal sequence
- it positions translational pause domain to interact with the elongation factor binding site
- the ribosome pauses in translation and the whole complex can be brought to ER where it interacts with SRP receptor
- this now gives appearance of rough ER
what happens once SRP binds to its receptor
it transfers the polypeptide to a protein translocator machinery (i.e. TOM), allowing protein to be threaded across ER membrane as its being translated
why is there no protein requirement for SRP
the process occurs before protein is folded
what generates a force to push a polypeptide out of a ribosome
adding a new amino acid to the polypeptide
what is the transmembrane region a stretch of
highly hydrophobic stretch of at least 8 amino acids
what happens to the transmembrane region as we’re synthesizing proteins
any time there is this long stretch, it gets stuck in membrane as transmembrane region
- some portions are pushed into ER, synthesized into cytosol, stuck in membrane as we push it back and forth across the membrane as many times is needed to have enough transmembrane regions
what happens if there is a single transmembrane region
we have signal sequence (start transfer sequence); thread polypeptide through until we reach another polypeptide stretch (stop transfer sequence - gets recognized and stuck in membrane); next hydrophobic would start transfer, and so on
what cleaves off signal peptide
signal peptidase enzyme - now N-terminus is in ER lumen; C-terminus is in cytosol; threading continues until C-terminus is in ER lumen
how do cells know where N-terminus and C-terminus should be in transmembrane regions
on either side of hydrophobic regions, first region that serves as start transfer has positive amino acid on one end and negatively charged amino acid at another.
- the side with the positively charged amino acid is going to face the cytosol (positive charge is n-terminus); negative charge faces lumen of ER
how do cells know where N-terminus and C-terminus should be in transmembrane regions if we have multi-pass polypeptide
hydrophobic regions = # transmembrane regions
- odd # regions = n terminus and c terminus are on opposite sides of ER membrane
- even # = will be on same side of membrane
what happens if positive charge is on n-terminal side
n-terminal will face cytosol
what happens if negative charge is towards n-terminal side
n-terminal faces lumen
