Figure 1: Ubiquitin-dependent proteolysis. Ubiquitin (Ub) is activated by covalent attachment to E1 enzyme in an ATP-dependent reaction and is subsequently transferred to an E2 (ubiquitin-conjugating) enzyme. Ubiquitin is transferred from E2 to target substrate with or without the assistance of an E3 (ubiquitin ligase) enzyme. E3s control the specificity and timing of ubiquitination reactions. After several cycles of ubiquitination, the multiubiquitin chain-bearing substrate is recognized by the 26S proteasome and degraded.
From the lab of Raymond Deshaies.
Figure 2: Control of cell division by ubiquitin-dependent proteolysis. SCF ubiquitin ligase enables the entry into S phase by promoting degradation of the S-phase cyclin-dependent kinase inhibitor Sic1. APC (anaphase-promoting complex) ubiquitin ligase enables chromosome segregation and exit from mitosis by promoting degradation of Pds1/securin and cyclin B (cycB), respectively.
From the lab of Raymond Deshaies.
Figure 3: Modular architecture of SCF and other cullin-RING ligases. Phosphorylated (P) Sic1 is recruited to SCF via the Cdc4 substrate receptor. Cdc4 is incorporated into SCF via its F-box domain (F), which binds Skp1. Skp1, in turn, binds the N-terminal domain of Cul1. The C-terminal domain of Cul1 binds the RING subunit, which recruits the E2 enzyme. In the example shown, E2 enzyme is thioesterified with a ubiquitin (Ub) molecule that will be transferred from E2 to substrate, as indicated by the arrow. N8 refers to the ubiquitin-like protein Nedd8, which is reversibly conjugated to the Cul1 subunit (see Figure 4).
From the lab of Raymond Deshaies.
Figure 4: Hypothetical model for a cullin cycle sustained by reversible modification with Nedd8. (This model incorporates CAND1, which is not discussed in the text.) CAND1 can only bind to Cul1 that is not modified with Nedd8. CAND1 and Skp1 bind Cul1 competitively. (1) Active CRL (cullin-RING ligase) is deneddylated by CSN (2). This may preferentially occur upon exhaustion of available substrate. Deneddylation enables the protein CAND1 to displace Skp1 from Cul1 (3), leading to complete sequestration of Cul1 by CAND1 (4). It is not certain how Cul1 is mobilized to reassemble into active SCF complexes. One possible scenario is that Ubc12, possibly with the assistance of an unknown factor X, conjugates Nedd8 upon Cul1 (5), which initiates CAND1 dissociation and enables binding of a Skp1–F-box protein (FBP) module (1). Alternatively, Skp1-FBP could displace CAND1, allowing for neddylation of Cul1 by Ubc12.
From Cope, G.A., and Deshaies, R.J. 2003. Cell 114:663–671. © 2003, with permission from Elsevier.
Figure 5: Strategy for identification of ubiquitin-proteasome system (UPS) ligands and substrates. To identify ubiquitin conjugates whose levels increase or decrease in a particular mutant or physiological state (in this case, a mutant lacking the proteasome substrate receptor Rpn10), wild-type cells are grown in medium formulated with "heavy" 15N nitrogen; mutant cells are grown in medium formulated with normal 14N nitrogen. Immediately prior to analysis, cells are mixed and lysed, and the cell extract is taken through a two-stage affinity purification to enrich specifically for ubiquitin conjugates destined for degradation by the proteasome. The resulting pool of conjugates is subjected to shotgun mass spectrometry to identify its component proteins. Ratiometric quantification of the 14N and 15N isotopomers for each peptide reveals the impact of rpn10D on the levels of all detectable ubiquitin conjugates. Up to 550 different ubiquitin conjugates can be identified and quantified in a 10-hour mass spectrometry analysis. In the example shown, ubiquitin conjugates of the "circle" protein accumulate in rpn10D cells, and thus the 14N/15N ratios for peptides derived from this protein are predicted to be >1. On the other hand, ubiquitin conjugates of the hexagonal protein and the square contaminant yield equivalent levels of 14N and 15N peptide isotopomers.
From the lab of Raymond Deshaies.
Figure 6: Protein phosphatase Cdc14 is released from the nucleolus during anaphase. Cdc14 (green) is localized to a discrete subnuclear domain (the nucleolus) during interphase, when cells contain either a single microtubule-organizing center or a short microtubule spindle (tubulin, red). In cells that have a long microtubule spindle indicative of late anaphase, Cdc14 is dispersed throughout the cell.
From the lab of Raymond Deshaies.
Figure 7: Control of the exit from mitosis. Net1 retains Cdc14 in an inactive form within the nucleolus. Upon activation of the mitotic exit network (MEN), Cdc14 is released from Net1, diffuses throughout the cell, and activates mitotic cyclin proteolysis and the exit from mitosis by dephosphorylating the APC regulator Hct1/Cdh1. It is not known how the activity of the mitotic exit network brings about activation of Cdc14.
From the lab of Raymond Deshaies.
