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)
