Targeting Proteins to Organelles Flashcards
Give an overview of protein targeting to organelles?
This is dictated by amino acid motifs
Evidence - shown through mutational analysis
Signals found on the final protein motif for organelle direction
Types: ER targeting signals Mitochondrial targeting signals Peroxisome targeting signals Nuclear import/export signals
Describe ER targeting signals?
Normal - signal recognition sequences
In order to retain proteins in the ER - KDEL receptor
The KDEL signal retrieves proteins that have trafficked to the Golgi - back to the ER in COPI vesicles
Example - BiP or PDI both contain KDEL, which is recognised by the KDEL receptor
How can the ER retrieval signals be masked?
ER retention signals
RKR and KKXX
Example: KATP channels and quality control
Masking retention motifs (e.g. RKR motifs) can permit ER export of ER retained proteins (e.g. KATP channels)
Give an overview of mitochondrial targeting signals?
These signals will aim to target the matrix - Matrix targeting signals
20-50 amino acids in length
Rich in hydrophobic amino acids (arginine and lysine)
Lack negatively charged amino acids (aspartate and glutamate)
Hydrophobic charges on one side of the helix, hydrophilic on the other: amphipathic
Mutation of these residues disrupts mitochondrial targeting
What is used in mitochondrial targeting?
Requires outer membrane receptors and translocons in both membrane
Import receptors: identified with antibodies that could block the mitochondrial translocation of proteins
Receptors recognise targeting sequences: Tom 20 and Tom 22 (Tom - translocation of the outer membrane)
Both are resident on the outer mitochondrial membrane
Molecular chaperones - HSP70 & HSP90
They use energy derived from ATP hydrolysis to keep proteins in a disaggregated state - available to be taken up by the mitochondria in an unfolded state
For some mitochondrial proteins, Tom70 serves as the import receptor through binding to HSP90
Describe receptor Tom40?
Import receptors transfer the precursor protein to an import channel in the outer mitochondrial membrane: Tom40
Tom40 - general import pore that is wide enough to accommodate an unfolded polypeptide chain
Tom40 pore is passive: driving force for import comes from the mitochondrial matrix
Describe Tim proteins?
Tim: Translocon of the Inner Membrane
Transfer through Tom40 is simultaneous with transfer through an inner membrane channel: Tim23 and Tim17
Translocation into the mitochondrial matrix occurs at contact sites at which the outer and inner membranes are in close proximity
Give a basic overview of the mechanism of mitochondrial targeting?
- ATP hydrolysis by HSP70: Cytosolic HSP70 expends energy maintaining the denatured polypeptide in an unfolded state
- ATP driven release of HSP70 from the translocating polypeptide: to “trap” it in the mitochondrial matrix
- H+ electrochemical gradient (proton motive force): Means that only mitochondria undergoing respiration can transfer precursor mitochondrial proteins
Describe the energy required for mitochondrial targeting?
- ATP hydrolysis by HSP70: Cytosolic HSP70 expends energy maintaining the denatured polypeptide in an unfolded state
- ATP driven release of HSP70 from the translocating polypeptide: to “trap” it in the mitochondrial matrix
- H+ electrochemical gradient (proton motive force): Means that only mitochondria undergoing respiration can transfer precursor mitochondrial proteins
Is there other mitochondrial targeting?
Mitochondrial targeting is not just the matrix
Targeting to the inner membrane, outer membrane and intermembrane space requires more than one targeting sequence and can occur via several pathways
What is involved in mitchondrial targeting - inner membrane proteins?
There are three main different pathways for this to take place - A, B and C
Mitochondrial targeting - inner membrane proteins - describe pathway A?
Pathway A
Same machinery as a matrix targeting protein - different recognition (stop/transfer)
N-terminal targeting sequence recognised by Tom20/22 which is transferred through Tom40 and the inner membrane Tim23/17
N-terminal sequence is cleaved
Contains a hydrophobic stop transfer sequence (anchor)
Translocation stops and the protein inserts laterally into the inner membrane, similar to an ER integral membrane protein
Mitochondrial targeting - inner membrane proteins - describe pathway B?
Pathway B
Contains a matrix targeted sequence and internal hydrophobic domain recognised by a protein Oxa1
N-terminal targeting sequence recognised by Tom20/22 which is transferred through Tom40 and the inner membrane Tim23/17
N-terminal sequence is cleaved
Hydrophobic domains are inserted into the membrane through Oxa1 interactions
Mitochondrial targeting - inner membrane proteins - describe pathway C?
Pathway C
Followed by proteins with ≥ 6 TM domains that lack the usual N-terminal matrix targeting sequence
Internal sequences are recognised Tom70 and Tom 22
Protein is translocated to the inner membrane Tim 22 & Tim 54
Transfer occurs through Tim9 and Tim10 that act as chaperones to stop protein folding/aggregation in the intermembrane space)
Tim22/54 insert each of the hydrophobic regions into the inner membrane
Mitochondrial targeting - inter membrane space?
There are two major pathways A and B
Pathway A
Proteins carry two signals
N-terminal matrix sequences are cleaved by the matrix protease
Contains a hydrophobic stop transfer sequence
Membrane sequence laterally diffuses from the Tim22/17 channel and is cleaved to release the protein into the intermembrane space
Example - haem binding to haem proteins
Pathway B
Proteins contain no N-terminal sequence matrix sequences
Delivered to the intermembrane space via the general import pore Tom40
No involvement of inner membrane factors
Disulphide bond formation (reminiscent to that in the ER lumen) through Mia40 and Erv1 (both disulphide bond generating proteins) “traps” the protein in the inner membrane space)
Describe mitochondrial targeting - outer membrane?
Outer mitochondrial membrane proteins (including Tom40) typically have a beta barrel arrangement
Proteins are incorporated onto the outer membrane through interactions with Tom40 and transfer to the sorting and assembly complex (SAM)
SAM complex consists of at least three proteins
Sam50 is closely related to bacterial BamA protein
SAM recognises pre-cursor complexes
BamA is responsible for the insertion of beta-barrel proteins into the membranes of gram negative bacteria
Precise mechanism of mitochondrial Sam insertion is still unknown
Give an overview of peroxisomes?
Small organelles bound by a single membrane
Unlike mitochondria no ribosome association
All luminal peroxisome proteins are synthesised on cytosolic ribosomes
Addition of new lipids and proteins leads to peroxisome division
Contain enzymes that use molecular oxygen to oxidise various substrates (amino acids and fatty acids)
H2O2 produced by the oxidation reactions are harmful to cellular components
Peroxisomes contain catalase that efficiently converts H2O2 to H2O
In mammals, peroxisomes are most abundant in liver cells
Describe peroxisomal targeting signals?
Governed by peroxisome targeting sequences
First discovered in luciferase: targeted to peroxisomes
C-terminal deletion led to loss of luciferase peroxisome targeting
Mutation of the C-terminus identified the sequence Ser-Lys-Leu (SKL)
Addition of the SKL signal to a non-peroxisome protein can target it to peroxisomes
Sequence is known as the Peroxisomal Targeting Sequence 1 (PST1)
Give an example of peroxisomal targeting of proteins?
Catalase
PST1 binds to a receptor: Pex5
Pex5 binds to the Pex14 receptor in the peroxisome membrane
Catalase is released from Pex14 into the interior of the peroxisome through the Pex2/10/12 complex
Translocates FOLDED proteins (unlike the ER and mitochondria)
PTS remains intact
Describe a peroxisomal defect?
Zellweger syndrome: congenital (absence of functional peroxisomes)
Transport of proteins into the peroxisome matrix is impaired
Pex5 mutations have been identified that fail to incorporate matrix proteins, producing empty peroxisomes that have the full complement of peroxisome membrane proteins
Thus membrane insertion is not impaired
Means that different proteins control the import of matrix proteins and the insertion of membrane proteins
Describe the mechanism of targeting membrane proteins to peroxisomes?
Insertion of membrane proteins occurs during peroxisome generation
- Biogenesis (begins in the ER). Pex3 and Pex16 are inserted into the ER membrane through co-translational translocation
- Pex3/16 recruit Pex19: specialized region of the ER that can bud off to form an empty peroxisome (pre-peroxisome)
- Complement of Pex proteins are recruited that permit protein import into the matrix.
Complete incorporation of matrix proteins yields a mature peroxisome
Describe nuclear targeting signals?
Entry and exit of large molecules from thenucleusis tightly controlled by nuclear pore complexes(NPCs)
Macromolecules such as RNA and proteins require association with transport factors to exit/enter the nucleus
Proteins to be imported: carrynuclear localization signals(NLS) that are recognised by importins
Incorporated in a folded state (unlike the ER)
NLS was discovered from large T-antigen of simian virus 40 (SV40)
Seven residue sequence rich in basic amino acids near the C-term
The ability of importins to transport their cargo is regulated by the small Ras relatedGTPase,Ran
Describe the nuclear localisation signal?
NES is a shortsequence of 4 hydrophobic residues
NES is recognized and bound byexportins
Common spacing of the hydrophobic residues - LxxxLxxLxL where “L” is a hydrophobic residue (often leucine) and “x” is any other amino acid
Exportin 1 recognizes the NES on Rev and the viral mRNA is subsequently transported by this complex from the nucleus through the nuclear pore to the cytoplasm
Translation of the viral message can then occur
Describe nuclear export signals?
Proteins also have nuclear export sequences (NES) - leucine-rich, short amino acid sequence of 4 hydrophobic residues
NES signal first discovered in the HIV-1 REV protein and cAMP-dependent protein kinase inhibitor (PKI)
Shuttle between nucleus and cytoplasm if contain both a NLS and NES
Describe protein targeting in plant cells?
“Although exceptions exist, a striking feature of the mechanisms and cellular machinery of protein targeting is their universality amongst plants, animals, and eukaryotic microorganisms”
There are exceptions such as the vacuole - as not present in mammalian cells
Most are the same e.g. SKL, KDEL etc…
