Sorting Flashcards
plasma membrane
outer boundary of cells
phospholipid bilayer
protection, transporters, cell signaling
nucleus
houses genome
RNA and DNA synthesis
cytoplasm
cytosol + cytoplasmic organelles
intermediary metabolism
endoplasmic reticulum
with ribosomes = rough
w/o = smooth
protein/lipid synthesis, protein folding, quality control, Ca storage, signaling
golgi apparatus
stacks of disc-like compartments
post-translational changes to proteins/lipids
trafficking
mitochondria
outer and inner membrane matrix
powerhouse of cell
signaling
cell differentiation and death
what happens when a mitochondria is leaky?
apoptosis
lysosomes
contain digestive enzymes to degrade organelles and biomolecules
peroxisomes
small vesicular compartments that contain enzymes used in oxidative rxns
topological compartments
- nucleus and cytosol
- secretory and endocytic organelles
- mitochondria
3 types of transport for trafficking
- gated transportation
- TM transportation
- vesicular trafficking
gated transportation
between nucleus and cytosol
thru nuclear pores
bidirectional
TM transportation
cytosol to peroxisomes, plastids, mito, ER
monodirectional
membrane transporters directly transport proteins from cytosol to target
vesicular trafficking
ER —-> elsewhere
use of membrane bound vesicles to transport molecules
what guides protein sorting?
protein sorting signals
protein sorting signals
sequence of AAs on protein
can be anywhere or multiple places in protein
but when folded they come together to form a signal patch
necessary and sufficient
signal peptidase
after a protein has reached it’s final destination
peptidase can cleave the signal sequence off because it is no longer needed
what is more important in a signal sequence?
physical properties are more important than the actual sequence
_____ receptors recognize and read signal sequences.
complimentary receptors
import into nucleus
lys and Arg rich
import into mitochondria
combination of + charged and hydrophobic AAs
import into ER
bunch of hydrophobic AAs
export from ER
KDEL
Lys-Asp-Glu-Leu-COO-
what molecules are exported from the nucleus?
mRNA and tRNA
NPC
nuclear pore complexes
NPC characteristics
composed of nucleoporins
octagonal
extending fibrils facilitate mvt
3000-4000 NPC per nucleus
NPC components
cytosolic fibers scaffold nucleoporins membrane ring proteins channel nucleoporins disordered region of C nucleoporins nuclear basket
NLS
nuclear localization signals
direct mvt of protein into nucleus
rich in + AAs (lys Arg)
located in loops or patches on surface of cargo
NIR
nuclear import receptors
cytosolic proteins that:
recognize NLS and bind to it and NPC proteins
types of NIR binding
direct binding
indirect binding
via adaptor protein
NPC binding sites for NIR
FG repeats
phenylalanine glycine repeats
after NIR delivers protein to destination where does it go?
delivers protein to nucleus and returns to cytosol
NES
nuclear export signals
same as import just signal opposite direction
NER
nuclear export receptors
complimentary to NES, bind to it and NPC proteins to move out of the nucleus
monomeric G protein
Ran
cytosolic Ran vs. nuclear Ran
GDP cytosol
GTP nucleus
Ran cytosol
GAP
GAPase activating protein
cleaves phosphate bond to keep Ran as GDP
Ran nucleus
GEF
guanine exchange factor
exchanges guanine with a GTP guanine
does not add Pi group
driving factor for gated transportation
Ran-location type gradient
Ran-GTP binding……
binds to (NIR + cargo) cargo is released NIR--Ran exit nucleus GAP cleaves Ran-GDP now ready for another cycle
proteins that contain both NLS and NES sequences
shuttling proteins
relative rate of gated transportation controls….
homeostasis = importing = exporting
import greater = nuclear
export greater = cytosolic
what controls transportation?
genes
keep proteins out of nucleus until they are needed
how is transportation controlled?
by turning NLS/NES on or off
mechanisms for transportation control?
phosphorylation
proteolysis
binding to inhibitory proteins
two examples given in this lecture: gated transportation
T-Cell activation
low cholesterol
mitochondria structures
outer memb.
intermemb. space
inner memb.
inner matrix
cristae of inner memb.
significance of cristae
folds in inner membrane function to increase surface area
source of mitochondria protein
most are encoded by nuclear DNA
but some are made in the mito itself, has it’s own translation machinery
translocation
mvt of proteins of membrane
what directs proteins to a specific organelle or compartment?
signal sequences
mitochondria signal sequences
located at N terminus or in middle
amphiphilic alpha helix shape
describe amphiphilic alpha helix
shape of proteins destined for mitochondria
created by positive residues on one end and hydrophobic ones on other end
nonpolar residues are ?
hydrophobic
how do receptor proteins recognize precursor proteins bound for mitochondria?
by the alpha helix configuration not the signal sequence itself
where are protein translocators located?
on the membrane of organelle protein is imported to
protein translocators of mitochondria
multi-subunit protein complexes that mediate translocation
list the mitochondrial translocators
cytosol to inter space
TOM
SAM
inter space to matrix
TIM 22
TIM 23
OXA
translocator that transfers or inserts all proteins from cytosol to outer membrane
TOM
sorting and assembly machinery
SAM
Translocates and inserts or folds beta barrel proteins
mediates insertion of specific subclass proteins
TIM22
ATP, ADP, Pi transporter
transport of soluble proteins and insertion of proteins into inner membrane
TIM23
insertion of proteins synthesized in the mitochondria
OXA
a few exceptions recently found
TOM and TIM have 2 components
receptors for precursor protein
translocation channel
describe a precursor protein
unfolded proteins in cytosol
maintained by chaperone hsp70
describe protein import through TOM
TOM binds to signal seq.
chaperones are stripped off –ATP
protein fed thru channel into space
peptidase cleaves signal
when is energy required in TM transport?
to remove hsp70
to remove mito hsp70 in matrix
for hsp60 to fold/refold protein in matrix
what drives protein mvt through TOM?
ATP hydrolysis drives removal of hsp70
free unfolded peptide is then pulled thru TOM
what drives protein mvt through TIM?
the electrochemical membrane potential gradient, drives the positive protein by electrophoresis
protein wants to get to the negatively charged matrix
mitochondrial hsp70
binds to protein in matrix and helps pull it thru TIM23
ATP is required for hsp70 to release
hsp60
binds to protein in mito matrix and helps fold the imported protein
requires ATP to do so
integration within the outer mitochondrial membrane
pass thru TOM
chaperones bind
protein binds to SAM
SAM - inserts and folds protein into memb.
example of TM proteins on outer mitochondrial membrane
porins
ER structure
network of branching tubules and sacs
membrane is continuous with nuclear memb.
internal space = ER lumen
types of ER translocation
co-translational
post-translational
co-translational translocation
mvt into ER
ribosome still attached
translation still in process
post-translational translocation
mvt into ER
translation finished
ER signal sequence
AA specific order varies
8 or more nonpolar/hydrophobic AA’s in center or protein
signal sequence guidance to ER—2 factors
SRP
SRP receptor
what does SRP stand for?
signal receptor particle
SRP structure
6 proteins bound to a small RNA backbone
rod shaped
large hydrophobic pocket
describe the hydrophobic pocket of SRPs
lined by methionines
accommodates hydrophobic signal seq. of varying size, shape, sequence
SRPs cycle back and forth between ?
cytosol and surface of ER membrane
what do SRPs bind to?
ER signal sequence on protein
SRP receptor of ER membrane
in co-translational: to large unit of ribosome
where does a SRP bind to a ribosome?
to the large unit of ribosome at the elongation factor
and binds to the ER signal sequence of the protein being translated
describe steps of co-translational translocation
SRP binds ribosome SRP binds ER signal seq. translation paused travel to ER bind to SRP receptor translation restarts protein fed thru translocator SRP recycled
where are the SRP receptors located?
next to translocators on ER membrane
describe ER translocators
circular shape
3 subunits
largest surrounds pore
central pore
describe ER translocator pore
water filled
core = Sec61 complex
gated by short helix
opens and closes as needed
what are the 2 states of the ER translocator?
open — full circle, pore plugged
closed — 3/4 circle shape, pore plug displaced
signal seq. bound in open 1/4
signal sequence that interacts with a specific site within the pore
start-transfer signal
also interacts with lipid components of ER membrane. acts as dual recognition to ensure specificity
what does the start-transfer signal do?
activates the pore opening
allowing entry to lumen
peptidase cleaves it off
integration of TM proteins to ER - requirements
some portion of the protein must pass thru the translocator before a stop-transfer signal is reached
what initiates integration of TM proteins in the ER?
N terminus
describe a stop transfer signal
a hydrophobic region in the polypeptide that stops translocation
peptidase cannot cleave because it becomes integrated into bilayer via the lateral gate of the translocator
can a TM protein be multi-integrated?
yes, single or multiple
depends upon the combination of start and stop transfer signals
in an ER TM protein which side of the membrane does each terminus of the protein end up on?
either side
they both can exist on the same side too
ER integration predictions
we can utilize software to predict the amount of integration of a protein
based upon the physical properties of the sequence itself
