The Steady-State Repertoire of Human SCF Ubiquitin Ligase Complexes Does Not Require Ongoing Nedd8 Conjugation

J Eugene Lee,Michael J Sweredoski,Robert L.J Graham,Sonja Hess,Geoffrey T Smith,Raymond J Deshaies,Natalie J Kolawa

doi:10.1074/mcp.m110.006460

Abstract

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.

Highlights

Proteins in the cell are in a dynamic state—they are continuously being synthesized and degraded to maintain intracellular protein homeostasis
Using the quantitative stable isotope labeling with amino acid in cell culture (SILAC) approach, we show that MLN4924 increased FBPSkp1 association with Cul1
We found at least 42 F-box proteins (FBPs) assembled with Cul1 in HEK 293 cells, with abundance of the complexes varying over two orders of magnitude

Summary

EXPERIMENTAL PROCEDURES

Chemicals and Reagents—MagneHis Ni-Particles and sequencing grade trypsin were purchased from Promega. Plasmid and Cloning—The HTBH tag [20] was a generous gift from the Kaiser group This tag contains a TEV cleavage site, two hexa-His sequences, and a biotinylation signal sequence. 100 ␮g/ml hygromycin was added to the medium to select for the cells with intergrated sequences coding for Cul1HTBH. Two-step Purification of Cul1HTBH—To induce the expression of HTBH-tagged Cul in cells at 50% confluency, 1.0 ␮g/ml tetracycline was added to the medium for 4 h. This expression condition ensured that the level of tagged Cul was similar to that of endogenous Cul. For emPAI value calculation, modified and shared peptides were included for counting

RESULTS

Number of observed peptides emPAId

Number of ratio measurementsd

DISCUSSION