| We have investigated different mitochondrial protein degradation pathways in the yeast, Saccharomyces cerevisiae, using mutationally altered forms of iso-1-cytochromes c (iso-1). Using these iso-1 substrates, we characterized different protein degradation pathways based on properties such as apo- or holo-forms, dependence or independence on ubiquitination or/and proteosome, global suppression by N52I, mitochondria rho+/rho- state, and suppression by different genetic modifiers. These pathways are (i) ubiquitin-dependent-degradation (UDD), (ii) rho-dependent-degradation (RDD), (iii) labile-dependent-degradation (LDD), and (iv) amphipathic-dependent-degradation (ADD).; In the ADD pathway, the N-terminal dispensable region of iso-1 was replaced by 11 different amphipathic structures, having various charge and hydrophobic moments. The N-terminal amphipathic structures diminished the iso-1 with an inverse correlation between the holo-1 levels and the amphipathic moments. Pulse chase experiments demonstrated that the amphipathic structures caused rapid iso-l degradation with the degree of degradation increasing with the amphipathic moments, but the apo forms were not affected. Iso-1 encoded by cyc1-868 has an N-terminal amphipathic structure with strongest first moments of charge and hydrophobicity, and resulted in a 10% steady state holo-iso-1 level and a 12-minutes half-life. We examined the effect of many genetic modifiers to understand the pathway involved in the rapid degradation of cyc1-868 iso-1. The degradation was suppressed by deletions of genes encoding mitochondria inner-membrane associated m- and i-AAA proteases, yme1-Delta, afg3-Delta or rca1-Delta. In contrast, cyc1-868 was not suppressed by deletion of mitochondrial matrix protease, mitochondrial inner-membrane chaperone proteins, various cytosolic ubiquitin conjugating enzymes, or by over expression of abnormal ubiquitin which prevents multi-ubiquitination. We suggest that amphipathic structures cause a stronger association with mitochondria inner membrane, and consequently with the inner-membrane associated proteases.; Furthermore, genetic analysis revealed that the ADD degradation is suppressed by the rho- condition in conjunction with certain chromosomal constitutions, Mitochondria-Dependent-Degradation (MDD), by mitochondria inhibitors CCCP and oligomycin, and by the global suppressor, N52I, which thermodynamically stabilizes iso-1. Mdd+ is a dominant phenotype; interestingly, Mdd- causes temperature sensitivity, which is suppressed by afg3-Delta or rca1-Delta.; Thus, we have identified and characterized a novel mitochondria pathway that acts on amphipathic structures, the ADD, adding to our knowledge of degradation of mitochondria protein. |
