INTERACTIONS AMONG 3 PROTEINS THAT SPECIFICALLY ACTIVATE TRANSLATION OF THE MITOCHONDRIAL COX3 MESSENGER-RNA IN SACCHAROMYCES-CEREVISIAE

The PET54, PET122, and PET494 proteins, which are associated with the yeast inner mitochondrial membrane, specifically activate translation of the mitochondrially encoded COX3 mRNA. We used the two-hybrid syste m to test whether pairs of these proteins, when fused to either the GA L4 DNA-binding or transcriptional activating domain, can physically as sociate as measured by the expression of the GAL4-dependent reporter, lacZ. PET54 and PET122 interacted in this system, and an amino-termina lly truncated PET494 fragment showed an interaction with PET54. We als o detected functional interactions between PET54 and PET122 geneticall y: a pet54 missense substitution (Phe and Gly at position 244) that ca used a severe respiratory defect was suppressed both by a missense sub stitution affecting PET122 (Gly to Val at position 211) and by overpro duction of wild-type PET122. Both Gly and Ala, substituted at PET54 po sition 244, disrupted the two-hybrid interactions with PET122 and PET4 94. While Ala at PET54 position 244 caused only a modest respiratory p henotype alone, it caused a severe respiratory defect when combined wi th a cold-sensitive mitochondrial mutation affecting the COX3 mRNA 5' leader. This synthetic defect was suppressed by a missense substitutio n in PET122 and by overproduction of wild-type PET122, indicating func tional interactions among PET54, PET122, and the mRNA. Taken together with previous work, these data suggest that a complex containing PET54 , PET122, and PET494 mediates the interaction of the COX3 mRNA with mi tochondrial ribosomes at the surface of the inner membrane.