We previously described the use of a differential hybridization screen of a genomic DNA library of Saccharomyces cerevisiae to identify sporulation-specific (SPS) genes (A. Percival-Smith and J. Segall, Mol. Cell. Biol. 4:142-150, 1984 logical events reflects the sequential expression of sporulation-specific genes. Although analyses of in vivo-labeled proteins by one-and two-dimensional gel electrophoresis revealed disappointingly few sporulation-specific proteins (17,20,31,38,45,46), mutational analyses identified a class of genes, termed SPO, dispensable for mitotic growth but essential for the completion of sporulation (10, 39). Expression of the SP013 gene has recently been shown to be developmentally regulated (41). In an alternative approach, genes expressed preferentially during sporulation have been directly identified by differential hybridization screening procedures (4, 13, 28). The transcripts encoded by these genes accumulate before the completion of meiosis (13,16,28). In complementary studies, analyses of the in vitro coding capacities of RNAs isolated from sporulating cells demonstrated the accumulation of an array of sporulationspecific mRNAs most of which also appear before the completion of meiosis (21,44). Two of the sporulationspecific transcripts identified in a study performed by Kurtz and Lindquist, however, begin to accumulate only after spore wall synthesis has begun (22). Interestingly, these two late transcripts, but not the sporulation-specific transcripts which accumulate earlier during meiosis, are present in mature asci (21). This observation prompted us to perform a differential hybridization screen of an S. cerevisiae DNA library using probes prepared with RNA from spores. As described in this report, this screen identified two sporulation-specific genes which are expressed very late during the sporulation process. A mutational analysis revealed that one of these genes contributes to spore wall maturation. 912The the on May 11, 2018 by guest

The endoplasmic reticulum of mammalian cells contains a heat shock protein of -70 kDa (hsp7O) termed binding protein BiP that is thought to promote the folding and subunit assembly of newly synthesized proteins. To study BiP function, we placed the BiP-encoding gene from Saccharomyces cerevisiae under the control of a regulated promoter and examined the effects of BiP depletion. Reduction of BiP protein to about 15% of normal levels led to a profound reduction in secretion of a factor and invertase. At the same time, unglycosylated precursors of these proteins accumulated intracellularly. The predominant form of the invertase precursor had undergone signal sequence cleavage but accumulated as a soluble species in the cytosol. In contrast, the a-factor precursor was exclusively in the signal-uncleaved form. It sedimented with microsomal membranes and was exposed at the cytoplasmic face in a protease-resistant form. These rmdings suggest that, in yeast, BiP function is required for translocation of soluble proteins into the endoplasmic reticulum at a stage beyond the initial nascent chain-membrane association.

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We previously described the use of a differential hybridization screen of a genomic DNA library of Saccharomyces cerevisiae to identify sporulation-specific (SPS) genes (A. Percival-Smith and J. Segall, Mol. Cell. Biol. 4:142-150, 1984). This initial screen identified 14 SPS genes that are first expressed 6 to 8 h after transfer of cells to sporulation medium. Accumulation of transcripts corresponding to these genes becomes maximal at 8 to 12 h of sporulation, the time at which meiotic events are nearing completion, and by 15 h of sporulation, transcript levels are beginning to decrease. In the present study two additional SPS genes, first expressed at 12 h of sporulation, were isolated. The steady-state level of transcripts corresponding to these two genes, termed SPS100 and SPS101, remains unchanged from 15 to 35 h, a time coincident with spore wall maturation. The nature of the putative 34.2-kilodalton protein encoded by the SPS100 gene is consistent with its being a component of the glycoprotein matrix of the spore wall; the protein contains a potential signal sequence and cleavage site and numerous sites for potential glycosylation. A MATa sps100/MAT alpha sps100 strain was found to be indistinguishable from the wild-type strain when assessed for efficiency of ascus formation and spore viability. However, a more detailed analysis of the mutant strain revealed that the SPS100 gene product serves a protective role during the early stages of spore wall formation. The time at which resistance to ether could first be detected in developing spores was delayed by 5 h in the mutant strain relative to the wild-type strain. This phenotype is presumably a reflection of a defect in spore wall maturation. This study has confirmed that temporally distinct classes of sporulation-specific genes are sequentially activated during the process of meiosis and spore formation and has shown that the SPS100 gene, identified on the basis of its developmental-specific expression pattern, contributes to spore development.