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.

SUMMARY The modular SCF ubiquitin ligases feature a large family of substrate receptors that enable recognition of diverse targets. However, how the repertoire of SCF complexes is sustained remains unclear. Real-time measurements of formation and disassembly indicate that SCFFbxw7 is extraordinarily stable but, in the Nedd8-deconjugated state, is rapidly disassembled by the cullin-binding protein Cand1. Binding and ubiquitylation assays show that Cand1 is a protein exchange factor that accelerates the rate at which Cul1–Rbx1 equilibrates with multiple F-box Protein–Skp1 modules. Depletion of Cand1 from cells impedes recruitment of new F-box proteins to pre-existing Cul1 and profoundly alters the cellular landscape of SCF complexes. We suggest that catalyzed protein exchange may be a general feature of dynamic macromolecular machines and propose a hypothesis for how substrates, Nedd8, and Cand1 collaborate to regulate the cellular repertoire of SCF complexes.

The methods used to perform the switching functions of the Bell System have been developed under the fundamental assumption that the holding time of the completed call is long compared to the time needed to set up the call. In considering certain forms of communication with and among computers the possibility arises that a message, with its destination at its head might thread its way through a communication network without awaiting the physical realization of a complete dedicated path before beginning on its journey. One such scheme has been proposed by J. R. Pierce and may be called “loop switching.” We imagine subscribers, perhaps best thought of as computer terminals or other data generating devices, on one‐way loops. These “local” loops are connected by various switching points to one another as well as to other “regional” loops which are in turn connected to one another as well as to a “national” loop. If a message from one loop is destined for a subscriber on another loop it proceeds around the originating loop to a suitable switching point where it may choose to enter a different loop, this process continuing until the message reaches its destination. The question naturally comes up, how the message is to know which sequence of loops to follow. It would be desirable for the equipment at each junction to be able to apply a simple test to the destination address at the head of the message which would determine which choice the message should make at that junction. In this paper we propose a method of addressing the loops which has several attractive features: It permits an extremely simple routing strategy to be used by the messages in reaching their destinations. By using this strategy, a message will always take the shortest possible path between any two local loops in the same region. The method of addressing applies to any collection of loops, no matter how complex their interconnections. The addressing scheme we propose will be applied primarily to local loops where the mutual interconnections may be quite varied. If a certain amount of hierarchical structure is introduced into the regional and national loop structure, as suggested by J. R. Pierce,1 it is possible to achieve addressings which are both compact and quite efficient.

Lens epithelium-derived growth factor (LEDGF/p75) tethers lentiviral preintegration complexes (PICs) to chromatin and is essential for effective HIV-1 replication. LEDGF/p75 interactions with lentiviral integrases are well characterized, but the structural basis for how LEDGF/p75 engages chromatin is unknown. We demonstrate that cellular LEDGF/p75 is tightly bound to mononucleosomes (MNs). Our proteomic experiments indicate that this interaction is direct and not mediated by other cellular factors. We determined the solution structure of LEDGF PWWP and monitored binding to the histone H3 tail containing trimethylated Lys36 (H3K36me3) and DNA by NMR. Results reveal two distinct functional interfaces of LEDGF PWWP: a well-defined hydrophobic cavity, which selectively interacts with the H3K36me3 peptide and adjacent basic surface, which non-specifically binds DNA. LEDGF PWWP exhibits nanomolar binding affinity to purified native MNs, but displays markedly lower affinities for the isolated H3K36me3 peptide and DNA. Furthermore, we show that LEDGF PWWP preferentially and tightly binds to in vitro reconstituted MNs containing a tri-methyl-lysine analogue at position 36 of H3 and not to their unmodified counterparts. We conclude that cooperative binding of the hydrophobic cavity and basic surface to the cognate histone peptide and DNA wrapped in MNs is essential for high-affinity binding to chromatin.