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.

At the onset of anaphase, a caspase-related protease (separase) destroys the link between sister chromatids by cleaving the cohesin subunit Scc1. During most of the cell cycle, separase is kept inactive by binding to an inhibitory protein called securin. Separase activation requires proteolysis of securin, which is mediated by an ubiquitin protein ligase called the anaphase-promoting complex. Cells regulate anaphase entry by delaying securin ubiquitination until all chromosomes have attached to the mitotic spindle. Though no longer regulated by this mitotic surveillance mechanism, sister separation remains tightly cell cycle regulated in yeast mutants lacking securin. We show here that the Polo/Cdc5 kinase phosphorylates serine residues adjacent to Scc1 cleavage sites and strongly enhances their cleavage. Phosphorylation of separase recognition sites may be highly conserved and regulates sister chromatid separation independently of securin.

Strain construction, materials, and Net1 mutagenesisAll strains used are in the W303 background (can1-100, leu2-3, his3-11, trp1-1, ura3-1, ade2-1) except where noted in the strain table (Supplementary Table 1). A strain expressing a stable form of Clb2 lacking both the KEN and destruction boxes (Clb2C 2 DK 100 )HA3 was used in over-expression experiments with Clb2 (1).Net1 mutant constructs were created as previously described (2). Briefly, a wild type NET1-myc9 epitope tagged construct was cloned into a modified pRS304 vector containing 300bp upstream of the ATG translation start site using NcoI and EagI. Sitedirected mutagenesis of Serine/Threonine to Alanine was carried out using QuikChange Site-Directed mutagenesis kit from Stratagene (La Jolla, CA). The indicated Serine/Threonine were mutated to Alanine in 169,212,231,252,259,356,362,384,385,497,611,676), 169,212,231,252,259),212,252), 166,212,252,297,304) where * indicates that residue 62 was mutated to ensure complete elimination of all Cdk consensus sites even though it was not determined to be phosphorylated in vivo. Mutagenesis was confirmed using restriction digests followed by Antibodies specifically reactive against the phosphopeptides were positively selected on a resin derivatized with the phosphopeptide immunogen and negatively selected by passage through a resin derivatized with the unphosphorylated version of the peptide. Antiphosphopeptide B (α-PP-B) was used in all experiments described since it generated the strongest signal against Phospho-Net1 ( Fig. 2A). Cell Growth and Synchronization ProceduresCells were grown in yeast extract-peptone (YP) or in yeast minimal (YM) media containing 2% glucose (YPD,YMD), 2% raffinose (YPR,YMR) or 2% galactose (YPG,YMG) as carbon source. Where appropriate, minimal media were supplemented with leucine, histidine, tryptophan, uracil, and adenine to complement auxotrophies.Synchronization of cells in G1 phase was achieved with α-factor added at 10 µg/ml for BAR1 cells and 0.1 µg/ml for bar1∆ cells for at least 3 hrs at 25°C. Cells were judged to be arrested when greater than 90% of cells displayed the elongated "shmoo" phenotype.Cells were released from α factor by filtration through a 0.2µm filter followed by a wash with 150 ml of YP, then resuspended in the desired volume at a density of 1 O.D. 600 /ml.For elutriation, cells were grown overnight in YP containing 2% raffinose and 2%galactose and harvested at log phase. Elutriation was performed as described (3-5) for the collection of small, unbudded G1 cells; contamination with budded cells was measured to be no more than 2%. For galactose induction experiments, cells were grown overnight in either YMR or YPR until an O.D. 600 of 1.0 was reached, then induced with 2% galactose followed by time point collection. Cell Extract Preparation and Western BlottingCells were grown to an O.D. 600 of 1.0, and for every time point 2 ml of culture was collected and TCA added to a final concentration of 20%. Cells were collected by centrifugation and washed with 2...