A mutational analysis reveals new functional interactions between domains of the Oxa1 protein in Saccharomyces cerevisiae.
The Oxa1/YidC/Alb3 family plays a key role in the biogenesis of the respiratory and photosynthetic complexes in bacteria and organelles. In Saccharomyces cerevisiae, Oxa1 mediates the co-translational insertion of mitochondrially encoded subunits of the three respiratory complexes III, IV and V within the inner membrane and also controls a late step in complex V assembly. No crystal structure of YidC or Oxa1 is available and little is known about the respective role of each transmembrane segment (TM) and hydrophilic loop of this polytopic protein on the biogenesis of the three complexes. Here, we have generated a collection of random point mutations located in the hydrophobic and hydrophilic domains of the protein and characterized their effects on the assembly of the three respiratory complexes. Our results show mutant-dependent differential effects, particularly on complex V. In order to identify tertiary interactions within Oxa1, we have also isolated revertants carrying second-site compensatory mutations able to restore respiration. This analysis reveals the existence of functional interactions between TM2 and TM5, TM4 and TM5 as well as between TM4 and loop 2, highlighting the key position of TM4 and TM5 in the Oxa1 protein.
Mathieu
Lise
L
Centre de Génétique Moléculaire du CNRS, FRE3144, FRC3115, Gif sur Yvette cedex, France.
Bourens
Myriam
M
Marsy
Sophie
S
Hlavacek
Otakar
O
Panozzo
Cristina
C
Dujardin
Geneviève
G
D019943:Amino Acid Substitution; D001665:Binding Sites; D004252:DNA Mutational Analysis / Q000379:methods*; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015151:Immunoblotting; D026901:Membrane Transport Proteins / Q000235:genetics; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010101:Oxygen Consumption / Q000235:genetics; D010641:Phenotype; D010788:Photosynthesis / Q000235:genetics; D014176:Protein Biosynthesis; D021122:Protein Subunits / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D026901:Membrane Transport Proteins; D024101:Mitochondrial Proteins; D009687:Nuclear Proteins; C090858:OXA1 protein; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D003576:Electron Transport Complex IV