Unit 6: Protein Sorting to Organelles Flashcards
Proteins must be:
Localized to the correct organelle.
Most proteins synthesized in eukaryotic cells
Are Encoded by nuclear DNA
Are synthesized on ribosomes (in cytosol)
Are delivered to the organelle (destination) from the cytosol
A few proteins synthesized in eukaryotic cells
Are encoded by the DNA in mitochondria and chloroplasts
Are synthesized on ribosomes inside mitochondria and chloroplasts
Are incorporated directly into compartments w/in mitochondria and chloroplasts
Definition of protein sorting and what allows it to be sorted to different organelles
The process by which newly-made proteins are directed to the correct location (organelles)
Amino acid sequence that signals where protein should go
Each protein has a _______________ which is often removed once protein arrives at it’s destination.
Sorting signal (signal sequence) of 3-60 continuous amino acids.
Retention of lumen of ER SS
(NH3+)…………..-K-D-E-L-(COO-)
For soluble membrane bound proteins
Import into ER SS
(NH3+)-M-M-(Lots of hydrophibic in a series, and then 3x -ve charged Glu (acidic) AA’s scattered)……..(COO-)
Import into Mitochondria SS
(NH3+)-M-(Lots of +ve charged (basic) AA’s scattered in long sequence)…….(COO-)
Import into nucleus SS
(NH3+)……..-P-P-K-K-K-R-K-V-…………(COO-)
Anywhere on sequence, 5x +ve charged (basic) amino acids in sequence
Import into peroxisomes
(NH3+)………-S-K-L-(COO-)
Polar neutral, +ve then hydrophobic
Difference between SS being NECESSARY and SUFFICIENT for protein sorting:
“Is this SS all that I need to signal it to get to where it needs to be (to get into the ER)?”
Separate ER SS from ER protein, does it still go into ER?
If you get rid of the SS and move the protein strand:
a) If it goes to another place… it is Necessary, but NOT Sufficient.
b) If it doesn’t go anywhere… it is Necessary AND Sufficient.
3 steps in protein sorting
- Recognition of the SS by a shuttling cytosolic receptor
- Targeting to the outer surface of the organelle membrane
- Import of the targeted protein into the membrane or transport of the protein across the membrane
A general problem for protein import into organelles is:
How to transport the protein across membranes that are normally impermeable to hydrophilic molecules?
Three main mechanisms to import proteins into a membrane-enclosed organelle
- Transport through nuclear pores
- Transport across membranes
- Transport by vesicles
Transport through nuclear pores (1 of 3) as a mechanism to import proteins into a membrane-enclosed organelle:
Transports SPECIFIC PROTEINS
Proteins remain FOLDED during transport
Transport across membranes (2 of 3) as a mechanism to import proteins into a membrane-enclosed organelle:
In the ER, mitochondria, chloroplasts, peroxisomes
Requires protein TRANSLOCATORS
Proteins are UNFOLDED in order to cross the membrane
Transport by vesicles (3 of 3) as a mechanism to import proteins into a membrane-enclosed organelle:
From ER, onward… and through EMS
Transport vesicles collect protein (cargo) and PINCH OFF from membrane
Deliver cargo by FUSING with another compartment
Proteins remain FOLDED during transport
Nuclear import general:
NPC (1000 molecules/second) both directions at the same time.
_____________ can move through NPC via passive diffusion.
Small, water-soluble molecules and proteins < 40kDA
NPC components
Fibrils
Membrane Ring Proteins
Channel Nucleoporins
Scaffold Nucleoporins
Cytosolic Fibrils
Fibrils location and function
Inside nucleus converge at distal ends, forming the nuclear basket. (not sure of function)
Membrane Ring Proteins function
Anchor NPC to nuclear envelope
Channel Nucleoporins function and location
Line the central pore, many unstructured regions containing F-G repeats, making up the mesh-like nature of NPC.
Scaffold Nucleoporins function
Membrane-bending, stabilize membrane curvature
Cytosolic Fibrils
Project outwards and helps direct cargo to NPC
Steps of nuclear import of proteins (mechanism)
- Proteins w/ NLS bind to NLS receptor (importin a/b heterodimer).
- The protein/importin complex associates w/ cytoplasmic filaments.
- The protein/importin complex passes through NPC…
- …..and associates w/ a GTPase (called RAN).
- The Ran●GTP-importin b complex is transported back to cytoplasm, where RAN is converted to Ran●GDP, brought back in to the nucleus. Importin a is returned to the cytoplasm via a protein called exportin.
Proteins synthesized in the cytoplasm are targeted for the nucleus by a ______, a common NLS is ________ with mostly _______ residues .
Nuclear Localization Signal (NLS),
P-K-K-K-R-K-V,
Basic AA residues (+ve charged)
Ran “gradient” ensures:
Directionality to nuclear transport. The GTP-bound (active) form only exists in the nucleus and the GDP-bound (inactive) form only exists in the cytosol.
Mitochondrial import general requirements
An NH3+ terminal (usually) SS required.
Only occurs at points where the inner and outer membranes are in close contact
In mitochondrial protein import, if the protein localizes to the intermembrane space, ____________.
A second sorting sequence is needed (matrix-targeting sequence)
Matrix-targeting sequences are:
Rich in hydrophobic, +ve charge and hydroxylated (Ser, Thr) AA residues, but have NO ACIDIC (-ve charged) AA residues.
M-T Sequences tend to form amphipathic helix.
Steps of Mitochondrial import mechanism (long version):
- Precursor proteins kept in unfolded state by action of the cytosolic chaperone (Hsc70), requires energy via ATP hydrolysis
- Then, M-T S interacts w/ receptor in outer mitochondrial membrane (TOM20 or TOM22)
- Receptor transfers the protein to the general import pore of the outer membrane (TOM40)
- / 5. At contact sites between the inner & outer membranes, the protein passes thru import pore of inner membrane (TIM23 & TIM17)
- Matrix (Hsc70) binds to (TIM44). ATP hydrolysis by this complex helps power translocation of protein into the matrix
- As M-T S emerges in the matrix, it is cleaved by a matrix Protease
- Protein can then fold into it’s final conformation, often (not always) assisted by matrix chaperonins.
Steps of Mitochondrial import mechanism (keyword version):
- Cytosolic Hsc70, ATP hydrolysis (energy)
- Receptor (TOM20 or TOM22)
- General import pore of outer membrane (TOM40)
- / 5. Import pore of the inner membrane (TIM23 and TIM17)
- Matric Hsc70 binds to TIM44. ATP hydrolysis (energy)
- Protease
- Fold
Additional requirement for protein import into mitochondria (coupling)
H+ electrochemical gradient generated by oxidative phosphorylation.
This ensures that only mitochondria that are actively respiring can import proteins.
When mitochondrial import mechanism is uncoupled with Oxidative phosphorylation
Blocks import of proteins into mitochondria
Notable details of mitochondrial import mechanism that targets proteins into the intermembrane space
Requires 2nd, hydrophobic targeting sequence that does not allow the protein to completely pass through the TIM23/17 import pore.
What does the 2nd hydrophobic targeting sequence that does not allow the protein to completely pass through the TIM23/17 import pore result in (targeting of proteins to the intermembrane space)
This stalled protein is then released from the pore into the membrane where a membrane- anchored protease cuts the protein, releasing it into the intermembrane space.
ER protein import requires
The ribosome that is synthesizing the protein to be attached to the ER membrane (~ rough ER lewk)
Unlike proteins that enter other organelles (Nucleus, Mitochondria, Chloroplasts, Peroxisomes) most proteins that enter ER:
Begin to be translocated (transported) across the ER membrane before the protein is completely synthesized.
There are two separate populations of ribosomes in the cytosol:
- Membrane-bound ribosomes
- Free ribosomes
Membrane-bound ribosomes are:
Attached to the cytosolic surface of the ER membrane and are synthesizing proteins that are translocated into the ER.
Free ribosomes are:
Unattached to any membrane (in cytosol) and are synthesizing all of the other proteins
Steps for importing a soluble protein into the ER lumen
- / 2. The emerging polypeptide with its ER signal sequence exposed is engaged by a complex of six proteins and an associated RNA molecule called the signal recognition particle (SRP). This binding halts translation and delivers the ribosome/polypeptide to the ER.
- SRP delivers the ribosome/polypeptide to the SRP receptor. This interaction is enhanced by the binding of GTP to both SRP and its receptor.
- The ribosome/polypeptide is then transferred to the translocon, inducing it to open and receive the polypeptide which enters as a loop. Hydrolysis of GTP by SRP and its receptor free these factors for another round of import.
- / 6. Translation then resumes and the signal sequence is cleaved by a membrane-bound protease called signal peptidase. Following this digestion, the rest of the protein is synthesized and enters the lumen of the ER
- / 8. Following completion of translation, the ribosome is released causing the translocon to close. The newly-synthesized protein then folds.
Six main types of membrane-anchored proteins:
- Type I
- Type II
- Type III
- Tail-anchored
- Type IV
- GPI-anchored
Membrane proteins of plasma membrane, golgi, lysosome and endosomes are all _________. They are then transported to their correct location using ___________.
Inserted into the ER membrane,
AA and carbohydrate sorting signals
Type I (Membrane Anchored Protein)
Is a single pass, cleaved SS at the NH3+ terminus, uses
SRP-SRP receptor to get to ER membrane.
Type II (Membrane Anchored Protein)
Single pass, no cleavable signal sequence, uses SRP-SRP
receptor to get to ER membrane, Nin-Cout
Type III (Membrane Anchored Protein)
same as type II, but Nout-Cin
Tail-anchored (Membrane Anchored Protein)
Single pass, no cleavable signal sequence, hydrophobic
membrane-spanning sequence at C-terminus, does not use SRP-SRP receptor but the GET1/2/3 system to get to ER, post-translational insertion, Nin-Cout
Type IV (Membrane Anchored Protein)
Multispanning, no cleavable signal sequence, uses SRP-SRP receptor for insertion of the first membrane-spanning domain but not subsequent ones, IV-A are Nin-Cin, IV-B are Nout-Cin
GPI-anchored (Membrane Anchored Protein)
Entire protein is lumenal (out), cleaved signal sequence at the N-terminus, uses SRP-SRP receptor to get to ER membrane, anchored at C-terminus to membrane and then transferred to GPI anchor.
Hydrophobic plots can help:
Determine the type of membrane protein. The hydropathic index for groups of 20 AA’s are calculated and plotted against the protein sequence.
More hydrophobic AA is along the sequence, the more +ve is the hydropathic index
More hydrophilic AA is along the sequence, the more -ve is the hydropathic index
Besides proteins residing in the ER, the ER is the starting point for:
- Soluble proteins that will be secreted from cell (hormones)
- Soluble proteins for Golgi, Lysosome or endosomes (acid hydrolases)
- Membrane proteins that will embed Golgi, lysosome, endosomes or plasma membrane (Na+/K+ ATPase)
In order to export the ER proteins, the ER ensures that they are:
Properly modified, folded and assembled by a process known as quality control.
Four principle modifications that occur in the ER (quality control):
- Disulfide bond formation
- Glycosylation
- Folding of polypeptides chains and assembly of multisubunit
complexes - Proteolytic cleavage of NH3+ terminal SS’s
ER quality control (1 of 4): Disulfide bond formation
B/w thiol groups of cysteine residues, on the same protein (intramolecular) or on two different proteins (intermolecular).
ER quality control (1 of 4): Disulfide bond formation dependent upon the enzyme
ER resident enzyme protein disulfide isomerase (PDI).
Consequences of ER resident enzyme PDI, only:
(i) secreted proteins or (ii) lumenal or extracellular domains of membrane proteins undergo this modification.
Disulfide bonds result in (effect) and what this is important for:
Stabilize protein structure
Important for proteins that will be subjected to extremes in pH/pOH or high protease environments.
ER quality control (2 of 4): Glycosylation
Begins w/ addition of a common oligosaccharide to the NH3+ group of Asn called N-linked glycosylation.
N-linked glycosylation
Addition of common oligosaccharide to Asn residues in the consensus sequence (Asn-X-Ser/Thr).
The precursor oligosaccharide in N-linked glycosylation is assembled in a ______ fashion on a _________
Step-wise,
Lipid molecule called Dolichol
The precursor oligosaccharide is transferred to the protein as the _______________. This requires an ER membrane – bound enzyme complex called ____________.
Consensus sequence emerges from the translocon
Oligosaccharyl transferase
Trimming in the ER and Golgi usually doesn’t involve the first
2x N-acetylglucosamine & 3x mannose attached to them.
In dolichol, the assembly of the 2 N-acetylglucosamine (GlcNAc) residues and first 5 mannose residues takes place on the________, which then flips (using a ______) to display oligosaccharide in the ____________.
Cytosolic surface of the ER,
transporter,
Lumen of the ER
After 7 first sugar residues added to oligosaccharide:
Remaining mannose and glucose residues added (one @ a time) until precursor is made
Attachment of sugars to dolichol is mediated by three main nucleotides:
- UDP-GlcNAc
- UDP-Glucose
- GDP-Mannose
What does tunicamycin do to formation of oligosaccharide precursor?
It blocks attachment of the 1st GlcNAc residue to dolichol
~ Non-glycosylated proteins
Why does tunicamycin result in Unfolded Protein Responses (UPR’s)?
Since glycosylation is used as a sign of protein folding and folding is required for export from the ER, this drug increases the level of unfolded proteins in the ER inducing the unfolded protein response (UPR).
Four roles of glycosylation:
- Promote folding of proteins
- Provide stability to proteins
- Promote cell-cell adhesion on plasma membrane proteins
- Act as a transport signal
Protein folding process and what assists in it
Molecular chaperones aid in protein folding by preventing aggregation of hydrophobic stretches of AA’s.
Two types of ER chaperones for protein folding:
- Classical chaperones
- Carbohydrate-binding chaperones
Classical ER chaperones (examples)
Hsp70 (BiP), Hsp90, GRP94
What do Carbohydrate-binding chaperones do, and give two examples:
Bind to monoglucosylated polypeptides.
The terminal glucose is removed.
If folded, the protein can exit the ER.
If not folded, a glucosyltransferase adds one glucose back
Then, cycle repeats.
Ultimately, if ________ are removed, the protein is targeted for ___________.
Mannose residues,
Dislocation (transport out of the ER) and degradation in cytosol.
