Lecture 7 - Protein Sorting Ch. 15 Flashcards
what does Cellular Compartmentalization allow for?
cellular specialization
what dominates cellular mass?
Proteins, which almost all synthesis begins in the cytosol
what is protein sorting?
The transfer of proteins from the cytosol to different organelles
Three fundamentally different mechanisms by which
proteins move from one compartment to another?
- Gated Transport
- transmembrane transport using protein translocators
- vesicular transport
where are 5 areas proteins go to begin the “roadmap”
nucleus, mitochondria, endoplasmic reticulum, peroxisomes, plastids(chloroplast)
where do all proteins eventually end up?
Cell exterior
how do the cytosolic proteins “know”
where to go in the cell?
- Proteins have specific amino acid sequences that direct them to their destinations.
- Sorting signals are recognized by receptors that guide proteins to the right location.
- Proteins without sorting signals stay in the cytosol.
If a protein has a signal sequence on the N-terminus where will the protein go?
Import to the ER or the mitochondria
What does recombinant DNA techniques do?
Between 2 proteins, it takes one of the signal sequence and it attaches it to the other protein. Both proteins will then go to their right location
Signal sequences are both ____ and ____ to direct a protein to a specific destination
necessary and sufficient
If a protein has a signal sequence on the C-terminus where will the protein go?
import into peroxisome or retention in lumen of ER
If a protein doesn’t have a signal sequence where will the protein go?
It will stay in the cytosol
Why do eukaryotic cells require a separate compartments?
a strategy to confine different metabolic processes and the proteins which are required for them for cellular specialization
Where can sorting signals be found in the amino acid sequence?
N-term or C-term
what usually happens to finished proteins?
The N-term is usually removed; other signal sequences become a part of the mature protein
Why must a sorting signal be distinct?
To allow selective recognition by the appropriate sorting receptor.
what is the first to be synthesized on polypeptide chain?
The N-term where synthesizes goes N -> C direction
where in the amino acid sequence of a protein is the signal sequence for import/export of the nucleus located?
internally within the amino acid (no N or C term)
What is KDEL, and what does it do?
KDEL is an ER retention signal, keeping proteins in the endoplasmic reticulum. It is named after its single-letter amino acid sequence: Lys-Asp-Glu-Leu
What are the internal signal sequences?
signal sequence is used to start the protein transfer, called a start-transfer sequence, is never removed from the polypeptide as an anchor
What type of sorting signals direct protein translocation into organelles?
Linear signal sequences guide proteins into organelles.
What is gated transport, and where does it occur?
A transport mechanism where molecules move between the nucleus and cytosol through nuclear pores.
What is the function of the lamin meshwork?
It provides structural support and serves as an anchoring site for nuclear pore complexes and chromosomes.
What is the composition of the nuclear pore complex (NPC) in animal cells?
molecular mass of 125 million Da, consists of 30 different nucleoporins in multiple copies, and contains a water-filled channel
How many NPCs does a typical mammalian cell have, and how do they function?
The nuclear envelope has 3000-4000 NPCs, transporting 1000 macromolecules per second in both directions simultaneously. NPCs are constitutively open.
Is nuclear import/export selective?
Yes,
Import: Histones, DNA polymerases, transcriptional regulators.
Export: mRNA, rRNA, tRNA, and other RNA molecules.
What is the size limit for passive diffusion through the NPC?
Diameter is ~40nm, allows small molecules (<5,000 Da) to diffuse freely. Molecules >40,000 Da require sorting signals for active transport. Larger molecules, like ribosomal subunits and some viruses, can pass through with assistance.
How do nuclear import receptors function?
Soluble cytosolic proteins that bind both the NLS on cargo proteins and NPC fibrils to mediate transport. Different receptors recognize different NLS signals. Nuclear proteins are transported fully folded, unlike ER/mitochondrial transport. Similar export receptors handle mRNA transport.
what protein triggers GTP hydrolysis located only in the cytosol?
Ran-GAP (GTPase-activating protein), converts Ran-GTP to Ran-GDP
What is Ran-GEF, and where is it located?
accessory protein Ran-GEF (guanine nucleotide exchange factor) is found only in nucleus & facilitates exchange of GDP for GTP on Ran (not substrate-level phosphorylation). This creates a higher concentration of Ran-GTP in the nucleus.
what is NLS?
nuclear localization signal
what are the 2 conformations of GTPase Ran?
One carrying GTP and the other carrying GDP
What is the kinase that phosphorylates Ran-GDP to Ran-GTP?
Ran does not undergo phosphorylation, conversion is done by GEF which exchanges GDP for GTP, which replaces the entire nucleoside
how do proteins transport to mitochondria and ER?
from cytosol through protein translocators (located in membrane) that must be unfolded
the matrix of the mitochondria?
high concentration of enzymes, fatty acid oxidation and citric acid cycle
Inner membrane of mitochondria?
folded cristae carries out oxidative phosphorylation. non permeable, requires transport across membrane
outer membrane of mitochondria?
contains porins (large channel-forming) render mitos permeable to molecules
intermembrane space of mitochondria?
site of proton buildup that drives ATP synthesis
how is mitochondria DNA synthesized?
encoded in the cell nucleus and imported from the cytosol
How do new mitochondria form and how are mitochondrial precursor proteins kept unfolded in the cytosol?
produced by the growth and fission of preexisting mitochondria, relying on protein import for expansion. Mitochondrial precursor proteins remain unfolded in the cytosol w/ the help of chaperone proteins. These include HSP70 family chaperones and proteins that bind directly to mitochondrial signal sequences to prevent premature folding.
chaperones aid in what?
helps fold proteins that are misfolded or newly synthesized.
mitochondrial proteins are first fully synthesized as….
precursor proteins in the cytosol
signal sequences that direct proteins into mitochondria has potential to form….
amphiphilic alpha helix
mitochondrial precursor proteins are imported as…..
unfolded polypeptide chains
how does the translocation into mitochondria work?
n-terminal sequence is recognized by transmembrane receptors, which is translocated through translocator through both membranes, signal sequence is cleaved and degrades in the matrix space
what occupies 10% of the total cell volume?
ER lumen and continuous sheet to the nuclear membrane
ER serves as….
entry point for proteins destined for other organelles
The ER membrane is the production site of all transmembrane proteins and lipids that include?
ER itself, Golgi, lysosomes, endosomes, secretary vesicles, and plasma membrane
majority of proteins that will be secreted to cell exterior…
are initially delivered to the ER lumen
what are the top 4 things that make up cell volume?
cytosol (54%), mitochondria (22%), ER (12%), Nucleus (6%)
what is the co-translational translocation process?
ribosome that is directly attached to ER membrane which enables one end of the protein to be translocated into ER while protein is still being assembled
what is post-translational translocation?
import of proteins into mitochondria and peroxisomes after synthesis of protein after translocation
what do free ribosomes in cytosol do?
allows for the mRNA encoding with no ER signal sequence, which remains in the cytosol
what do membrane-bound ribosomes attached to the ER do?
Has a ER signal sequence which directs the ribosome to the ER membrane where the protein is fed into the mitochondria
What 2 components is needed to direct ribosome to the ER membrane?
Signal recognition particle (SRP) in the cytosol and SRP receptor embedded in the ER membrane
what two proteins are synthesized by membrane-bound ribosomes?
water-soluble proteins are completely translocated across the ER membrane and prospective transmembrane proteins which are partly translocated across the membrane and become embedded into it
how does the water soluble protein translocate across the ER membrane
translocator binds to the signal sequence, where is cleaved by signal peptidase. This is ejected into bilayer where it degrades. The protein translocator closes once the polypeptide is release in the ER lumen.
proteins that remain in the ER have a what?
C-terminus KDEL signal sequence
how does prospective transmembrane proteins translocate across the ER membrane?
