Parkin, an E3 ubiquitin ligase implicated in Parkinson's disease, promotes degradation of dysfunctional mitochondria by autophagy. Using proteomic and cellular approaches, we show that upon translocation to mitochondria, Parkin activates the ubiquitin–proteasome system (UPS) for widespread degradation of outer membrane proteins. This is evidenced by an increase in K48-linked polyubiquitin on mitochondria, recruitment of the 26S proteasome and rapid degradation of multiple outer membrane proteins. The degradation of proteins by the UPS occurs independently of the autophagy pathway, and inhibition of the 26S proteasome completely abrogates Parkin-mediated mitophagy in HeLa, SH-SY5Y and mouse cells. Although the mitofusins Mfn1 and Mfn2 are rapid degradation targets of Parkin, we find that degradation of additional targets is essential for mitophagy. These results indicate that remodeling of the mitochondrial outer membrane proteome is important for mitophagy, and reveal a causal link between the UPS and autophagy, the major pathways for degradation of intracellular substrates.

The sorting and assembly machinery (SAM) complex functions in the assembly of beta-barrel proteins into the mitochondrial outer membrane. It is related to the Omp85/YaeT machinery in bacterial outer membranes, but the eukaryotic SAM complex is distinguished by two peripheral subunits, Sam37 and Sam35, that sit on the cytosolic face of the complex. The function of these subunits in beta-barrel protein assembly is currently unclear. By screening a library of sam35 mutants, we show that 13 distinct alleles were each specifically suppressed by overexpression of SAM37. Two of these mutants, sam35-409 and sam35-424, show distinct phenotypes that enable us to distinguish the function of Sam35 from that of Sam37. Sam35 is required for the SAM complex to bind outer membrane substrate proteins: destabilization of Sam35 inhibits substrate binding by Sam50. Sam37 acts later than Sam35, apparently to assist release of substrates from the SAM complex. Very different environments surround bacteria and mitochondria, and we discuss the role of Sam35 and Sam37 in terms of the problems peculiar to mitochondrial protein substrates.

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Mitochondria were derived from endosymbiotic bacteria and the mitochondrial outer membrane shares many features with bacterial outer membranes [1,2]. Polypeptides embedded in the outer membrane of Gram-negative bacteria are either lipoproteins anchored by a covalently linked lipid, or have a b-barrel structure with eight or more antiparallel b-strands hydrogen bonded into a cylindrical barrel [3,4]. The b-barrel proteins have an unusual primary structure with many strands of alternating hydrophilic and hydrophobic residues and a high abundance of aromatic residues that tend to be placed at the start of the strands [2,5]. These b-barrel proteins are assembled in the bacterial outer membrane in a process mediated by the integral membrane protein Omp85 [2,3,[5][6][7][8].Mitochondria also carry a member of the Omp85 family [8]: this protein, called Sam50, has been shown responsible for the assembly of b-barrel proteins in the mitochondrial outer membrane [9][10][11], and functions together with at least two other subunits as part of a Sorting and Assembly Machine (SAM) complex [7,[9][10][11]. In addition to b-barrel proteins, mitochondrial outer membranes also have proteins with a-helical transmembrane domains. These proteins appear to provide functions that were procured after the initial endosymbiont established itself in early eukaryotic cells, including protein Mitochondria evolved from a bacterial endosymbiont ancestor in which the integral outer membrane proteins would have been b-barrel structured within the plane of the membrane. Initial proteomics on the outer membrane from yeast mitochondria suggest that while most of the protein components are integral in the membrane, most of these mitochondrial proteins behave as if they have a-helical transmembrane domains, rather than b-barrels. These proteins are usually predicted to have a single a-helical transmembrane segment at either the N-or C-terminus, however, more complex topologies are also seen. We purified the novel outer membrane protein Om14 and show it is encoded in the gene YBR230c. Protein sequencing revealed an intron is spliced from the transcript, and both transcription from the YBR230c gene and steady-state level of the Om14 protein is dramatically less in cells grown on glucose than in cells grown on nonfermentable carbon sources. Hydropathy predictions together with data from limited protease digestion show three a-helical transmembrane segments in Om14. The a-helical outer membrane proteins provide functions derived after the endosymbiotic event, and require the translocase in the outer mitochondrial membrane complex for insertion into the outer membrane.Abbreviations DAS, dense alignment surface; PVDF, poly(vinylidene difluoride); TOM, translocase in the outer mitochondrial membrane.