Correspondence A novel family of predicted retroviral-like aspartyl proteases with a possible key role in eukaryotic cell cycle control

A yeast gene, DDI1, has been identified as a suppressor of a temperature sensitive mutant of the PDS1 gene, which encodes Pds1p (Securin), a key regulator of the cell cycle whose ubiquitin-dependent proteolysis initiates anaphase in budding yeast [1–3]. The Ddi1 protein has, at its carboxyl terminus, a ubiquitin associated domain that is required for the suppression of Pds1 mutations and has been shown to interact with ubiquitin [1,4]. Ddi1p is conserved in all eukaryotes for which sequence information is available, including the early-branching Protozoans Leishmania and Plasmodium, suggesting that this protein has an ancient, critical function. The biochemical activity of Ddi1p and its orthologs, however, remains unknown. Here we show that Ddi1p contains a conserved domain homologous to retroviral aspartyl proteases and propose that the protease activity of this protein is involved in a novel, ubiquitin-dependent branch of cell cycle control that operates in all eukaryotes, possibly as a previously undetected deubiquitinating enzyme. During a systematic analysis of eukaryotic Clusters of Orthologous Groups (COG) of proteins ([5,6] DMK, RL Tatusov, and EVK, unpublished), we identified the Ddi1p COG as one of the relatively small number of COGs with a high R584 Current Biology Vol 11 No 15 level of sequence conservation, conserved domain organization of most members, and representation throughout all eukaryotic genomes. Examination of the multiple alignment of the Ddi1p orthologs, which was constructed using the Clustal_X program [7], revealed the conservation in all these sequences of an aspartate–serine/threonine–glycine, D[ST]G signature, which is a hallmark of aspartyl proteases [8]. This observation prompted a more detailed analysis of the Ddi1p family protein sequences. A PSI-BLAST search [9] against the non-redundant (NR) protein sequence database at the National Center for Biotechnology Information (NIH, Bethesda), seeded with residues 205–296 of Ddi1p, including the DTG signature and roughly the size of an aspartyl protease domain, revealed statistically significant similarity to aspartyl protease domains of several retrotransposable elements. A reciprocal search started with the HIV aspartyl protease domain sequence also showed significant similarity to Ddi1p (see Figure 1 for details). In addition to the Ddi1p orthologs, these searches identified a group of related, predicted mammalian aspartyl proteases typified by the mouse neuron specific nuclear receptor interacting protein NIX1 [10]. Given the high conservation of Ddi1p orthologs in eukaryotes we were interested in detecting potential prokaryotic homologs of these proteins. A search of the prokaryotic protein sequences from the NR database with the sequence …