A proteomic screen reveals the mitochondrial outer membrane protein Mdm34p as an essential target of the F-box protein Mdm30p.
Ubiquitination plays various critical roles in eukaryotic cellular regulation and is mediated by a cascade of enzymes including ubiquitin protein ligase (E3). The Skp1-Cullin-F-box protein complex comprises the largest E3 family, in each member of which a unique F-box protein binds its targets to define substrate specificity. Although genome sequencing uncovers a growing number of F-box proteins, most of them have remained as "orphans" because of the difficulties in identification of their substrates. To address this issue, we tested a quantitative proteomic approach by combining the stable isotope labeling by amino acids in cell culture (SILAC), parallel affinity purification (PAP) that we had developed for efficient enrichment of ubiquitinated proteins, and mass spectrometry (MS). We applied this SILAC-PAP-MS approach to compare ubiquitinated proteins between yeast cells with and without over-expressed Mdm30p, an F-box protein implicated in mitochondrial morphology. Consequently, we identified the mitochondrial outer membrane protein Mdm34p as a target of Mdm30p. Furthermore, we found that mitochondrial defects induced by deletion of MDM30 are not only recapitulated by a mutant Mdm34p defective in interaction with Mdm30p but alleviated by ubiquitination-mimicking forms of Mdm34p. These results indicate that Mdm34p is a physiologically important target of Mdm30p.
Ota
Kazuhisa
K
Department of Computational Biology and Center for Omics and Bioinformatics, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa 277-8561, Japan.
Kito
Keiji
K
Okada
Satoshi
S
Ito
Takashi
T
D003470:Culture Media; D044783:F-Box Proteins / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism
D003470:Culture Media; D044783:F-Box Proteins; D024101:Mitochondrial Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin