Parkin uses the UPS to ship off dysfunctional mitochondria

Parkin is a ubiquitin E3 ligase that is implicated in familial Parkinson disease (PD). Previous studies have established its role in mitophagy, a pathway whereby dysfunctional mitochondria are targeted for autophagic degradation. We recently reported that a major function of Parkin in dysfunctional mitochondria is to activate the ubiquitin-proteasome system (UPS) for proteolysis of multiple outer membrane proteins, and that such activation of the UPS is a critical step in Parkin-mediated mitophagy. Here, we discuss the possible roles of the UPS in mitophagy and the pathogenesis of PD

Degradation of the Deubiquitinating Enzyme USP33 is Mediated by p97 and the Ubiquitin Ligase HERC2

Because the deubiquitinating enzyme USP33 is involved in several important cellular processes (β-adrenergic receptor recycling, centrosome amplification, RalB signaling, and cancer cell migration), its levels must be carefully regulated. Using quantitative mass spectrometry, we found that the intracellular level of USP33 is highly sensitive to the activity of p97. Knockdown or chemical inhibition of p97 causes robust accumulation of USP33 due to inhibition of its degradation. The p97 adaptor complex involved in this function is the Ufd1-Npl4 heterodimer. Furthermore, we identified HERC2, a HECT-domain-containing E3 ligase, as responsible for polyubiquitination of USP33. Inhibition of p97 causes accumulation of polyubiquitinated USP33, suggesting that p97 is required for post-ubiquitination processing. Thus, our study has identified several key molecules that control USP33 degradation within the ubiquitin-proteasome system

Broad activation of the ubiquitin-proteasome system by Parkin is critical for mitophagy

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

Evidence of a Reduced and Modified Mitochondrial Protein Import Apparatus in Microsporidian Mitosomes▿

Microsporidia are a group of highly adapted obligate intracellular parasites that are now recognized as close relatives of fungi. Their adaptation to parasitism has resulted in broad and severe reduction at (i) a genomic level by extensive gene loss, gene compaction, and gene shortening; (ii) a biochemical level with the loss of much basic metabolism; and (iii) a cellular level, resulting in lost or cryptic organelles. Consistent with this trend, the mitochondrion is severely reduced, lacking ATP synthesis and other typical functions and apparently containing only a fraction of the proteins of canonical mitochondria. We have investigated the mitochondrial protein import apparatus of this reduced organelle in the microsporidian Encephalitozoon cuniculi and find evidence of reduced and modified machinery. Notably, a putative outer membrane receptor, Tom70, is reduced in length but maintains a conserved structure chiefly consisting of tetratricopeptide repeats. When expressed in Saccharomyces cerevisiae, EcTom70 inserts with the correct topology into the outer membrane of mitochondria but is unable to complement the growth defects of Tom70-deficient yeast. We have scanned genomic data using hidden Markov models for other homologues of import machinery proteins and find evidence of severe reduction of this system

The Peripheral Membrane Subunits of the SAM Complex Function Codependently in Mitochondrial Outer Membrane Biogenesis

The sorting and assembly machinery (SAM) complex functions in the assembly of β-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 β-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