Spindle alignment is the process in which the two spindle poles are directed toward preselected and opposite cell ends. In budding yeast, the APC-related molecule Kar9 is required for proper alignment of the spindle with the mother-bud axis. We find that Kar9 localizes to the prospective daughter cell spindle pole. Kar9 is transferred from the pole to cytoplasmic microtubules, which are then guided in a myosin-dependent manner to the bud. Clb4/Cdc28 kinase phosphorylates Kar9 and accumulates on the pole destined to the mother cell. Mutations that block phosphorylation at Cdc28 consensus sites result in localization of Kar9 to both poles and target them both to the bud. Thus, Clb4/Cdc28 prevents Kar9 loading on the mother bound pole. In turn, asymmetric distribution of Kar9 ensures that only one pole orients toward the bud. Our results indicate that Cdk1-dependent spindle asymmetry ensures proper alignment of the mitotic spindle with the cell division axis.

Ubiquitin conjugation is known to target protein substrates primarily to degradation by the proteasome or via the endocytic route. Here we describe a novel protein modification pathway in yeast which mediates the conjugation of RUB1, a ubiquitin-like protein displaying 53% amino acid identity to ubiquitin. We show that RUB1 conjugation requires at least three proteins in vivo. ULA1 and UBA3 are related to the N-and C-terminal domains of the E1 ubiquitinactivating enzyme, respectively, and together fulfil E1-like functions for RUB1 activation. RUB1 conjugation also requires UBC12, a protein related to E2 ubiquitinconjugating enzymes, which functions analogously to E2 enzymes in RUB1-protein conjugate formation. Conjugation of RUB1 is not essential for normal cell growth and appears to be selective for a small set of substrates. Remarkably, CDC53/cullin, a common subunit of the multifunctional SCF ubiquitin ligase, was found to be a major substrate for RUB1 conjugation. This suggests that the RUB1 conjugation pathway is functionally affiliated to the ubiquitinproteasome system and may play a regulatory role.

The ubiquitin-like protein SMT3 from Saccharomyces cerevisiae and SUMO-1, its mammalian homolog, can be covalently attached to other proteins posttranslationally. Conjugation of ubiquitin requires the activities of ubiquitin-activating (E1) and -conjugating (E2) enzymes and proceeds via thioester-linked enzyme-ubiquitin intermediates. Herein we show that UBC9, one of the 13 different E2 enzymes from yeast, is required for SMT3 conjugation in vivo. Moreover, recombinant yeast and mammalian UBC9 enzymes were found to form thioester complexes with SMT3 and SUMO-1, respectively. This suggests that UBC9 functions as an E2 in a SMT3͞SUMO-1 conjugation pathway analogous to ubiquitinconjugating enzymes. The role of yeast UBC9 in cell cycle progression may thus be mediated through its SMT3 conjugation activity.Ubiquitin, a highly conserved protein of 76 amino acid residues, can be attached to other proteins posttranslationally (for reviews, see refs. 1-3). This reaction involves the formation of an isopeptide bond between the carboxyl-terminal glycine residue of ubiquitin and the -amino group of a lysine residue of an acceptor protein. All known functions of ubiquitin, including its role in selective protein degradation, are thought to be mediated through this reaction. Conjugation of ubiquitin proceeds via a reaction cascade involving ubiquitin-activating (E1) and ubiquitin-conjugating (E2) enzymes and, at least in some cases, ubiquitin-protein ligases (E3). The E1 enzyme hydrolyzes ATP and forms a high-energy thioester intermediate between a cysteine residue of its active site and the carboxyl terminus of ubiquitin. Ubiquitin is then passed on to E2 enzymes that form thioesters with ubiquitin in a similar fashion. Finally, ubiquitin is covalently attached to a substrate protein by the E2 enzymes or, alternatively, by E3 enzymes as indicated by the observation that at least some E3 enzymes are loaded with ubiquitin by E2 enzymes via thioester formation (4).The yeast Saccharomyces cerevisiae genome contains several homologous genes encoding putative E1 enzymes (UBA genes). UBA1 is essential for viability and encodes the activating enzyme required for ubiquitin conjugation (5). UBA2, also required for viability, encodes a smaller protein that bears a cysteine residue at a position similar to the active-site cysteine of UBA1 (6). A uba2 mutant, expressing a protein that lacks this cysteine, is inviable, suggesting that this conserved residue is involved in thioester formation as well. Surprisingly, however, purified UBA2 failed to form a thioester complex with ubiquitin, suggesting that it may function in a different pathway (6). Indeed, UBA2 was recently shown to cooperate with another protein termed AOS1 in the activation of SMT3, a protein of 98 amino acid residues with 17% sequence similarity to ubiquitin (7). Interestingly, AOS1, also encoded by an essential gene, and UBA2 show sequence similarity to the amino-and the carboxyl-terminal domains of the UBA1 protein, respectively. This suggests that upon heterodi...