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.

p97 is an ATP-dependent chaperone that plays an important role in endoplasmic reticulum-associated degradation but whose connections to turnover of soluble proteins remain sparse. Binding of p97 to substrates is mediated by cofactors that contain ubiquitin-binding domains. We employed "network proteomics" to show that p97 assembles with all of the 13 mammalian UBX-domain proteins. The UBX proteins that bind ubiquitin conjugates also interact with dozens of E3 ubiquitin ligases, only one of which had been previously linked to p97. In particular, UBXD7 links p97 to the ubiquitin ligase CUL2/VHL and its substrate hypoxia-inducible factor 1alpha (HIF1alpha). Depletion of p97 leads to accumulation of endogenous HIF1alpha and increased expression of a HIF1alpha target gene. The large number of ubiquitin ligases found associated with UBX proteins suggests that p97 plays a far broader role than previously anticipated in the global regulation of protein turnover.

Ubiquitin-dependent proteolysis can initiate at ribosomes for myriad reasons including misfolding of a nascent chain or stalling of the ribosome during translation of mRNA. Clearance of a stalled complex is required to recycle the ribosome for future use. Here we show that the ubiquitin (Ub) pathway segregase Cdc48/p97 and its adaptors Ufd1-Npl4 participate in ribosome-associated degradation (RAD) by mediating the clearance of ubiquitinated, tRNA-linked nascent peptides from ribosomes. Through characterization of both endogenously-generated and heterologous model substrates for the RAD pathway, we conclude that budding yeast Cdc48 functions downstream of the Ub ligases Ltn1 and Ubr1 to release nascent proteins from the ribosome so that they can be degraded by the proteasome. Defective RAD could contribute to the pathophysiology of human diseases caused by mutations in p97.DOI: http://dx.doi.org/10.7554/eLife.00308.001

BackgroundThe proteasome is a multi-subunit protein machine that is the final destination for cellular proteins that have been marked for degradation via an ubiquitin (Ub) chain appendage. These ubiquitylated proteins either bind directly to the intrinsic proteasome ubiqutin chain receptors Rpn10, Rpn13, or Rpt5, or are shuttled to the proteasome by Rad23, Dsk2, or Ddi1. The latter proteins share an Ub association domain (UBA) for binding poly-Ub chains and an Ub-like-domain (UBL) for binding to the proteasome. It has been proposed that shuttling receptors dock on the proteasome via Rpn1, but the precise nature of the docking site remains poorly defined.ResultsTo shed light on the recruitment of shuttling receptors to the proteasome, we performed both site-directed mutagenesis and genetic screening to identify mutations in Rpn1 that disrupt its binding to UBA-UBL proteins. Here we demonstrate that delivery of Ub conjugates and docking of Ddi1 (and to a lesser extent Dsk2) to the proteasome are strongly impaired by an aspartic acid to alanine point mutation in the highly-conserved D517 residue of Rpn1. Moreover, degradation of the Ddi1-dependent proteasome substrate, Ufo1, is blocked in rpn1-D517A yeast cells. By contrast, Rad23 recruitment to the proteasome is not affected by rpn1-D517A.ConclusionsThese studies provide insight into the mechanism by which the UBA-UBL protein Ddi1 is recruited to the proteasome to enable Ub-dependent degradation of its ligands. Our studies suggest that different UBA-UBL proteins are recruited to the proteasome by distinct mechanisms.

The human genome encodes 69 different F-box proteins (FBPs), each of which can potentially assemble with Skp1-Cul1-RING to serve as the substrate specificity subunit of an SCF ubiquitin ligase complex. SCF activity is switched on by conjugation of the ubiquitin-like protein Nedd8 to Cul1. Cycles of Nedd8 conjugation and deconjugation acting in conjunction with the Cul1-sequestering factor Cand1 are thought to control dynamic cycles of SCF assembly and disassembly, which would enable a dynamic equilibrium between the Cul1-RING catalytic core of SCF and the cellular repertoire of FBPs. To test this hypothesis, we determined the cellular composition of SCF complexes and evaluated the impact of Nedd8 conjugation on this steady-state. At least 42 FBPs assembled with Cul1 in HEK 293 cells, and the levels of Cul1-bound FBPs varied by over two orders of magnitude. Unexpectedly, quantitative mass spectrometry revealed that blockade of Nedd8 conjugation led to a modest increase, rather than a decrease, in the overall level of most SCF complexes. We suggest that multiple mechanisms including FBP dissociation and turnover cooperate to maintain the cellular pool of SCF ubiquitin ligases.