JAMM: A Metalloprotease-Like Zinc Site in the Proteasome and Signalosome
Figure 4
Mutations in the JAMM Motif of Csn5 Abrogate the Deneddylating Activity of the CSN
(A) Mutations in the glutamic acid (E56A) that positions the aqua ligand and in the proposed catalytic serine (S128A) of Csn5 disrupt deneddylation of Cul1 by CSN but have no effect on assembly with Csn1. A csn5Δ strain of S. pombe was transformed with an empty pREP-41 plasmid (lane 1) or with the plasmid encoding FLAG tagged: Csn5 (lane 2), Csn5E56A (lane 3), or Csn5S128A (lane 4). Whole-cell lysates were used for Western blot analysis with anti-Cul1 antibodies (top gel) and anti-FLAG antibodies (second from top). A strain with a myc13-tagged Csn1 was transformed with the above plasmids, and whole-cell lysates were used for Western blot analysis. Antibodies to the Myc tag were used to detect Csn1myc13 (third from top), and were used to pull down Csn1myc13 and subsequently blot with anti-FLAG antibodies to detect coprecipitated Csn5 mutant proteins (bottom gel).
(B) Mutations in the JAMM motif display a modest dominant-negative phenotype. Western blot analysis of crude cell lysates was performed as described in (A).
(C) Selected JAMM motifs from proteins of diverse functions. The canonical JAMM motif residues are highlighted in green. The conserved proline is highlighted in blue, and semiconserved cysteine is highlighted in yellow.