Protein secretion and sorting Flashcards
What are organelles and how do they originate?
functionally distinct, membrane-bounded compartments.
First only DNA and membrane bound organelles like in PROKARYOTIC CELLS. DNA surrounded by nucleus gaining a membrane
Mitochondria initially an external prokaryotic cell that incorporates eukaryotic cell membrane.
Name three transport mechanisms for proteins
1)Gated transport
occurs between cytosol and nucleus
2)Transmembrane transport
occurs where organelles distinct, ie from cytosol to ER
3)Vesicular transport
occurs between systems like ER to golgi to vesicles ect.
How are proteins sorted/sent to different regions in the cell?
Proteins have SIGNAL sequences or signal PATCHES where there separate regions on the unfolded peptide that interact (become the patch) when folded.
Describe difference between signal peptides and patches AFTER sorting.
Signal peptides: 15-60 aa long, often removed by specialized signal peptidases .
Signal patches remain part of the protein with a selfgovering
Function.
What role do Nuclear Pores (NPC) have?
Proteins enter nucleus through nuclear pores.
Small proteins (< 5 kDa) diffuse through the pores.
Large proteins (> 5 kDa) require targeting sequences and an active mechanism
What is the NPC and what is its structure?
It is a large protein complex (octagonal) containing over 30 different protein nucleoporins forming a channel for protein import and export
There is about 3000in one cell.
Describe how Proteins are transported through the nuclear pore?
protein contains a NLS (Nuclear Localisation Sequence) often found interal in its structure. ie, this does not get removed.
NLS recogised by an importin receptor known as KARYOPHERIN (there are specific importins)
Exportin receptors mediate protein and mRNA out of nucleus.
What is Ran in terms of the nuclear pore?
What is GAP and GEF and what does it do here?
Ran is a GTPase - a small GTP-binding protein with GTP hydrolysis activity.
Its gradient is important to active transport across the nuclear membrane; energy is produced by the hydrolysis of GTP
Its concentration is HIGH in the NUCLEUS
LOW in the CYTOPLASM.
GAP (GTPase activating enzyme) and GEF (Guanine exchange factor)
They trigger the Conversion between the stages RanGTP and RanGDP
What machinery is used to transport proteins into the mitochondria?
By a twin-pore translocator
Membrane has two membranes and two compartments: intermembrane and the matrix spaces.
TOM receptor on mitochondrial outer membrane
(~9 membrane proteins in a complex)
recognises signal sequence and acts to translocate protein to the inter-membrane space.
TIM complexes (on the inner mitochondrial membrane) TIM17-23 complex translocates proteins to the matrix
TIM22-54 complex translocates proteins to the inner membrane
Proteins passed DIRECTLY from TOM to TIM
after import Protein signal at the N (18-25) terminal is rapidly cleaved by the matrix processing protease (signal peptidase)
What is a Amphipathic alpha-helical helix?
cluster of positively helix where there are charged aa
on one side and hydrophobic cluster on the opposite side;
Describe the energy gradients involved in protein mitochondria transport.
1) First,in the cytoplasm, Hsp70chaperones on the importing protein are taken off by ATP hydrolysis (ATP made).
2) Next an Electrochemical H+ gradient is used to drive translocation through the TIM
3) Finally, Hsp chaperones IN THE MT are taken off the imported protein by hydrolysis of ATP (making ADP)
What determines which proteins end up where in the Mt?
Normally, proteins taken to matrix.
but Proteins residing in the inter-membrane space or the inner membrane such as CYTOCHROME C1, require multipart signals with additional information
CYTOCHROME C1 has 61 amino acid leader:
first 32 specify matrix localisation when fused to other proteins (uncharged/basic)
The next 19 amino acids target protein to remain in its proper location inner membrane (uncharged/hydrophobic)
Describe the process of protein translocation secretion involving the ER.
This involves Budding and fusion of membrane-bound vesicles.
Translocation across the ER membrane is through a GATED, protein-lined pore or translocon which is mediated by a SRP (SIGNAL RECOGNITION PARTICLE)
The N terminal leader sequence o the protein is made of HYDROPHOBIC residues.
After the proteins translocation through the membrane, the signal is cleaved.
Describe the E.R.
It is a netlike web of branched tubules
Its has an important role in lipid and protein formation; almost all proteins are processed through the E.R
There are ribosomes on Rough E.R surface.
Step by step of the process of protein secretion involving the ER.
1) Ribosome starts protein synthesis
2) SRP attaches to new proteins hydrophobic, n terminal leader (12-20) sequence and stops translation at about 70 aa.
3) SRP binds to its receptor, then ribosome attaches to the E.R membrane and translation starts again.
4) Leader sequence enters the membrane and the protein follows.
5) Leader sequence is cleaved
6) translation continued until entire protein is secreted.
7) ribosomes released from mRNA.
What is the SRP in E.R secretion?
Signal Recognition Particle is a ribonucleoprotein complex of 7S RNA + 6 proteins that binds to the proteins signal sequence and then to a SRP receptor on the E.R membrane.
Its conformation changes when it binds to the signal as it not only binds to the signal (at SRP54) but bends to also bind to the ribosome (SRP19-ELGONGATION FACTOR) the protein is emerging from.
What is the Translocon in E.R secretion?
Translocon is a pore in the membrane of the E.R containing a channel.
Consists of Sec61 core (alpha, beta and gamma subunits) with gates on both sides.
There is a gate on both sides.
Ribosome seals from cytoplasm side with SRP.
Internal E.R side opens when protein synthesis is resumed.
Protein goes through channel from cytoplasm into lumen of E.R
List types of membrane proteins
1) Peripheral- not embedded but have ionic interactions with protein/lipid head groups.
2) Integral-inserted into membrane and can be transmembrane.
3) Lipid and GPI anchored proteins
The TMD of a new protein gains access to the lipid bilayer through a TRANSLOCON channel formed by the helixes of the TMD
What is a Transmembrane domain (TMD)?
TMD adopts an α-helical conformation
A helix of 21-26 hydrophobic/non-polar amino acids that can ‘DISSOLVE’ in the lipid membrane.
R groups (usually hydrophobic aa) face out into the lipid while the Hydrogen bonded AMIDE bonds face inside the helix.
Different TMDs may prefer different surrounding lipids.
Describe two types of transmembrane protein.
Type 1: (C-terminus on cytosolic side) signal peptide cleaved but transfer stops at a second hydrophobic N domain (“stop transfer” sequence) that becomes the TMD.
Type II: (N-terminus on the cytosolic side) signal peptide remains membrane-associated but orientation of polypeptide is flipped during the translocation process (“signal anchor”)
The more POSITIVE end of the TMD is on the INSIDE CYTOPLASM whether it is type 1 or 2 or not.
What is a MULTI transmembrane protein
Proteins with multiple TMD, the number of which will determine the orientation of the protein.
If there is an EVEN number of TMD, both N and C ends will be on the same side.
UNEVEN number of TMD will lead to N end being on the opposite side from C.
Give an example of a single TM protein.
Glycophorin:131 AA long.
It consists of an ectodomain (with the N terminal), transmembrane domain (23 aa long) and a cytoplasmic domain (with the C terminal)
I is a TYPE 1 TMD protein as the N termal is OUTSIDE.
It is very GLYCOSYLATED
What is a Multi-pass TM protein
One protein structures span the bilayer several times (several strands) where there be POLAR and charged aa.
TMD may be doughnut shape forming a pore with these POLAR aa facing eachother; channels formed that can transport polar molecules (glucose)
Give an example of a multipass protein.
Bacteriorhodopsin; consists of 7TM alpha helices.
Type 1: as N terminus OUTSIDE membrane.
