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Although polyubiquitin chains linked through all lysines of ubiquitin exist, specific functions are well-established only for lysine-48 and lysine-63 linkages in Saccharomyces cerevisiae. To uncover pathways regulated by distinct linkages, genetic interactions between a gene deletion library and a panel of lysine-to-arginine ubiquitin mutants were systematically identified. The K11R mutant had strong genetic interactions with threonine biosynthetic genes. Consistently, we found that K11R mutants import threonine poorly. The K11R mutant also exhibited a strong genetic interaction with a subunit of the anaphase-promoting complex (APC), suggesting a role in cell cycle regulation. K11-linkages are important for vertebrate APC function, but this was not previously described in yeast. We show that the yeast APC also modifies substrates with K11-linkages in vitro, and that those chains contribute to normal APC-substrate turnover in vivo. This study reveals comprehensive genetic interactomes of polyubiquitin chains and characterizes the role of K11-chains in two biological pathways.

The Candida albicans orthologue of the SPC3 gene, which encodes one of the subunits essential for the activity of the signal peptidase complex in Saccharomyces cerevisiae, was isolated by complementation of a thermosensitive mutation in the S. cerevisiae SEC61 gene. The cloned gene (CaSPC3 ) encodes a putative protein of 192 amino acids that contains one potential membrane-spanning region and shares significant homology with the corresponding products from mammalian (Spc22/23p) and yeast (Spc3p) cells. CaSPC3 is essential for cell viability, since a hemizygous strain containing a single copy of CaSPC3 under control of the methionine-repressible MET3 promoter did not grow in the presence of methionine and cysteine. The cloned gene could rescue the phenotype associated with a spc3 mutation in S. cerevisiae, indicating that it is the true C. albicans orthologue of SPC3. However, in contrast with results previously described for its S. cerevisiae orthologue, CaSPC3 was not able to complement the thermosensitive growth associated with a mutation in the SEC11 gene. The heterologous complementation of the sec61 mutant suggests that Spc3p could play a role in the interaction that it is known to occur between the translocon (Sec61 complex) and the signal peptidase complex, at the endoplasmic reticulum membrane.

The Pichia anomala LEU2 gene (PaLEU2) was isolated by complementation of a leu2 Saccharomyces cerevisiae mutant. The cloned gene also allowed growth of a Escherichia coli leuB mutant in leucine-lacking medium, indicating that it encodes a product able to complement the b-isopropylmalate dehydrogenase deficiency of the mutants. The sequenced DNA fragment contains a complete ORF of 1092 bp, and the deduced polypeptide shares significant homologies with the products of the LEU2 genes from S. cerevisiae (84% identity) and other yeast species. A sequence resembling the GC-rich palindrome motif identified in the 5k region of S. cerevisiae LEU2 gene as the binding site for the transcription activating factor encoded by the LEU3 gene was found at the promoter region. In addition, upstream of the PaLEU2 the 3k-terminal half of a gene of the same orientation, encoding a homologue of the S. cerevisiae NFS1/SPL1 gene that encodes a mitochondrial cysteine desulphurase involved in both tRNA processing and mitochondrial metabolism, was found. The genomic organization of the PaNFS1-PaLEU2 gene pair is similar to that found in several other yeast species, including S. cerevisiae and Candida albicans, except that in some of them the LEU2 gene appears in the reverse orientation. The nucleotide sequence has been submitted to the EMBL database under Accession No. AJ294714.

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