Yeast Hct1 recognizes the mitotic cyclin Clb2 and other substrates of the ubiquitin ligase APC

Abstract

Ubiquitin‐mediated proteolysis has emerged as a key mechanism of regulation in eukaryotic cells. During cell division, a multi‐subunit ubiquitin ligase termed the anaphase promoting complex (APC) targets critical regulatory proteins such as securin and mitotic cyclins, and thereby triggers chromosome separation and exit from mitosis. Previous studies in the yeast Saccharomyces cerevisiae identified the conserved WD40 proteins Cdc20 and Hct1 (Cdh1) as substrate‐specific activators of the APC, but their precise mechanism of action has remained unclear. This study provides evidence that Hct1 functions as a substrate receptor that recognizes target proteins and recruits them to the APC for ubiquitylation and subsequent proteolysis. By co‐immunoprecipitation, we found that Hct1 interacted with the mitotic cyclins Clb2 and Clb3 and the polo‐related kinase Cdc5, whereas Cdc20 interacted with the securin Pds1. Failure to interact with Hct1 resulted in stabilization of Clb2. Analysis of Hct1 derivatives identified the C‐box, a motif required for APC association of Hct1 and conserved among Cdc20‐related proteins. We propose that proteins of the Cdc20 family are substrate recognition subunits of the ubiquitin ligase APC.

The anaphase-promoting complex: a key factor in the regulation of cell cycle

“…A second study demonstrated that the APC substrate, Pds1, whose degradation is exclusively induced by APC Cdc20 , binds directly Cdc20, whereas it is incapable of associating with Cdh1/Hct1, indicating that Cdc20 and Cdh1/Hct1 recognize and bind their substrates differently and selectively (Hilioti et al, 2001). Further specific and direct interactions of Cdc20 with Pds1 and of Cdh1/Hct1 with Clb2, Clb3 and Cdc5 were also demonstrated in budding yeast (Burton and Solomon, 2001;Schwab et al, 2001). Finally, Pfleger et al (2001a) showed that in the absence of APC, Cdc20 and Cdh1/Hct1 can directly and specifically bind a large number of APC substrates including Xkid, securin, geminin, cyclin A and cyclin B.…”

“…This interaction seems to be mediated through direct binding of the TPR subunits Apc3/Cdc27 and Apc7 with the IR C-terminal tails of Cdc20 and Cdh1/Hct1 (see above) . In addition to the IR motif, another sequence in the N-terminus of these activators, the C-Box, has indeed been found necessary for their association with APC in yeast (Schwab et al, 2001). Thus, further studies are required to establish the real mechanism through which these proteins bind the APC.…”

Loss of the anaphase-promoting complex in quiescent cells causes unscheduled hepatocyte proliferation

“…Whereas Cdc20 is active during mitosis, Cdh1 is active during G 1 , when Cdk1 is inactive. Phosphorylation of Cdh1 by Cdk1 prevents it from binding the APC/C for much of the cell cycle (Schwab et al 2001). The activities of Cdk1 and APC-Cdh1 are therefore mutually exclusive.…”

Understanding the structural basis for controlling chromosome division

“…Apc10 and coactivator have completely different three-dimensional structures, but both share a conserved C-terminal Ile-Arg (IR) motif required for binding to the tetratricopeptide repeat (TPR) subunit Cdc27/Apc3 [19][20][21][22][23]. In addition to the IR tail, coactivators interact with the APC/C through a C box motif within their N-terminal regions [24]. The C box has been shown to stimulate the catalytic activity of the APC/C [25].…”