Apg13p and Vac8p are part of a complex of phosphoproteins that are required for cytoplasm to vacuole targeting.

We have been studying protein components that function in the cytoplasm to vacuole targeting (Cvt) pathway and the overlapping process of macroautophagy. The Vac8 and Apg13 proteins are required for the import of aminopeptidase I (API) through the Cvt pathway. We have identified a protein-protein interaction between Vac8p and Apg13p by both two-hybrid and co-immunoprecipitation analysis. Subcellular fractionation of API indicates that Vac8p and Apg13p are involved in the vesicle formation step of the Cvt pathway. Kinetic analysis of the Cvt pathway and autophagy indicates that, although Vac8p is essential for Cvt transport, it is less important for autophagy. In vivo phosphorylation experiments demonstrate that both Vac8p and Apg13p are phosphorylated proteins, and Apg13p phosphorylation is regulated by changing nutrient conditions. Although Apg13p interacts with the serine/threonine kinase Apg1p, this protein is not required for phosphorylation of either Vac8p or Apg13p. Subcellular fractionation experiments indicate that Apg13p and a fraction of Apg1p are membrane-associated. Vac8p and Apg13p may be part of a larger protein complex that includes Apg1p and additional interacting proteins. Together, these components may form a protein complex that regulates the conversion between Cvt transport and autophagy in response to changing nutrient conditions.

Examination of the intron in the meiosis-specific recombination gene REC114 in Saccharomyces.

REC114 is one of 10 genes known to be required for the initiation of meiotic recombination in Saccharomyces cerevisiae. It is transcribed only in meiosis, and our previous sequence analysis suggested the presence of an intron in the 3' end of the gene. Hypotheses in the literature have suggested, because of its unusual location, either that the putative intron in REC114 is likely to be necessary for expression, or that there may actually be no intron present. This work demonstrates that REC114 does have an intron and is one of only three genes in yeast with introns located in the 3' end. Furthermore, the 3' splice site utilized in REC114 is a very rare AAG sequence; only three other genes in yeast use this nonconsensus sequence. The splicing of REC114 does not require MER1, a gene known to be involved in meiosis-specific RNA processing. In fact, an intronless copy of REC114 can complement a null rec114 mutation. Thus, it does not appear that the intron is essential for expression of REC114. Although the intron is not absolutely required for meiotic function, it is conserved in evolution; two other species of yeast contain an intron at the same location in their REC114 genes.

Recombination and the progression of meiosis in Saccharomyces cerevisiae.

Recombination is an essential part of meiosis: in almost all organisms, including Saccharomyces cerevisiae, proper chromosome segregation and the viability of meiotic products is dependent upon normal levels of recombination. In this article we examine the kinetics of the meiotic divisions in four mutants defective in the initiation of recombination. We find that mutations in any of three Early Exchange genes (REC104, REC114 or REC102) confer a phenotype in which the reductional division occurs earlier than in an isogenic wild-type diploid. We also present data confirming previous reports that strains with a mutation in the Early Exchange gene. MEI4 undergo the first division at about the same time as wild-type cells. The rec104 mutation is epistatic to the mei4 mutation for the timing of the first division. These observations suggest a possible relationship between the initiation of recombination and the timing of the reductional division. These data also allow these four Early Exchange genes examined to be distinguished in terms of their role in coordinating recombination with the reductional division.

Isolation of early meiotic recombination genes analogous to S. cerevisiae REC104 from the yeasts S. paradoxus and S. pastorianus.

The REC104 gene of Saccharomyces cerevisiae is required to initiate recombination in meiosis. Mutations in REC104 eliminate meiotic recombination and lead to the production of inviable spores. To determine if analogous genes exist in other yeasts, clones that hybridized to a REC104 probe were isolated from the yeasts S. paradoxus and S. pastorianus. When transformed into a rec104 strain, the REC104 analogs from these two yeasts restored spore viability and meiotic recombination to the same level as a REC104 gene cloned from S. cerevisiae. Compared to S. cerevisiae, the S. paradoxus gene codes for 79% identical amino acids and has 86% nucleic-acid identity in the promoter region and 84% in the coding region. The S. pastorianus gene codes for 63% identical amino acids and has 59% and 71% identity in the promoter and the coding regions, respectively.