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Mitochondrial Mas70p signal anchor sequence. Mutations in the transmembrane domain that disrupt dimerization but not
1Department of Biochemistry, McGill University, Montreal, Canada.
Abstract:
Mas70p is an integral membrane protein in Saccharomyces cerevisiae that is targeted and inserted into the mitochondrial outer membrane in an N(in)-Ccyto orientation by its NH2-terminal 29-amino acid signal anchor sequence. Recently, we demonstrated that the signal anchor was capable of mediating homo-oligomerization of a fusion protein, pOMD29, in the outer membrane in vitro (Millar, D. G., and Shore, G. C. (1992) J. Biol. Chem. 268, 18403-18406). Consistent with this finding, we show here that a synthetic peptide corresponding to the Mas70p signal anchor is capable of independent membrane insertion and dimerization with pOMD29. To further map the oligomerization domain in the signal anchor sequence, a deletion mutant of pOMD29 that lacks amino acids 2-10 was constructed. This protein, pOMD29 delta 2-10, efficiently participated in dimer formation following import, indicating that dimerization was mediated by the putative membrane spanning segment (amino acids 11-29). This segment is predicted to form an alpha-helix that has an alanine-rich face and contains multiple copies of a pentapeptide dimerization motif that is widespread among members of the receptor tyrosine kinase family. Substitution of the alanine residues in one of these copies with isoleucine, producing a potentially bulkier contact surface, resulted in a protein which was targeted and inserted into the outer membrane but failed to assemble into dimers. Taken together, these results identify a structural feature of the signal anchor transmembrane domain that is important for oligomerization but is not required for targeting and membrane insertion.
Insights
The Mas70p signal anchor sequence in yeast mitochondria mediates protein oligomerization. A specific transmembrane segment, rich in alanine, is crucial for this dimerization, independent of membrane targeting.
Area of Science:
- Cell Biology
- Molecular Biology
- Protein Trafficking
Background:
- Mas70p is an integral membrane protein in Saccharomyces cerevisiae.
- Its N-terminal signal anchor sequence directs insertion into the mitochondrial outer membrane.
- Previous work showed this anchor mediates fusion protein homo-oligomerization.
Purpose of the Study:
- To investigate the role of the Mas70p signal anchor sequence in protein dimerization.
- To map the specific domain responsible for oligomerization within the signal anchor.
- To determine the structural requirements for dimerization independent of membrane targeting.
Main Methods:
- Used synthetic peptides and deletion mutants of a Mas70p fusion protein (pOMD29).
- Assessed membrane insertion and dimerization in vitro and following import.
- Employed site-directed mutagenesis to alter specific residues within the putative dimerization domain.
Main Results:
- A synthetic Mas70p signal anchor peptide inserted into membranes and dimerized with pOMD29.
- A deletion mutant lacking amino acids 2-10 still formed dimers, implicating the membrane-spanning segment (aa 11-29).
- Mutating alanine residues in a pentapeptide motif within the transmembrane segment abolished dimerization, despite successful membrane insertion.
Conclusions:
- The transmembrane segment of the Mas70p signal anchor contains a dimerization domain.
- An alanine-rich face within this segment is critical for mediating protein oligomerization.
- This structural feature is essential for dimerization but not for initial membrane targeting and insertion.