Towards understanding cell cycle control in Cryptococcus neoformans: structure-function relationship of G1 and G1/S cyclins homologue CnCln1.

We have previously reported that only a single Cdk1-related G1 and G1/S cyclin homologue was found in the genome sequence of the pathogenic basidiomycetous yeast Cryptococcus neoformans (C. neoformans) and designated it CnCln1. Surprisingly, CnCln1 was not only able to complement the function of the G1 cyclins of the ascomycetous budding yeast Saccharomyces cerevisiae (S. cerevisiae), such as ScCln3, but also the G1/S cyclins of S. cerevisiae, such as ScCln1 and ScCln2. In this study, we investigated how CnCln1 cooperates with the cyclin-dependent kinases of S. cerevisiae (ScCdk1) and substitutes the function of G1 and G1/S cyclins of S. cerevisia from a point of view of their structure-function relationship. Our in silico analysis demonstrated that the CnCln1/ScCdk1 complex was more stable than any of the yeast cyclin and ScCdk1complexes. Thus, these results are consistent with in vitro analysis that has revealed the flexible functional capacity of CnCln1 as a Cdk1-related G1 and G1/S cyclins of S. cerevisiae.

Scanning and negative-staining electron microscopy of protoplast regeneration of a wild-type and two chitin synthase mutants in the pathogenic yeast Candida glabrata.

Protoplast regeneration of a wild-type and two mutant strains of Candida glabrata defective in CHS3 homologues encoding class IV chitin synthase in Saccharomyces cerevisiae was examined by scanning and negative-staining electron microscopy. In the wild-type strain, small particles and short filaments appeared on the protoplast surface at 10 min, filamentous materials covered the entire surface of the protoplast at 1 h, granular materials started filling interspaces of filamentous materials at 2 h and regeneration was completed at 6 h. The filamentous materials consisted of microfibrils of various widths ranging from ≤5 to 40 nm, and composed of ß-glucan. Protoplasts of the two chitin synthase mutant strains of Δchs3A and Δchs3B completed regeneration essentially by the same process as wild-type strain, although it took more time. These results suggest that CHS3A and CHS3B genes may have important roles in cell wall formation during protoplast regeneration, but can be compensated by other cell wall enzymes.

The single Cdk1-G1 cyclin of Cryptococcus neoformans is not essential for cell cycle progression, but plays important roles in the proper commitment to DNA synthesis and bud emergence in this yeast.

The cell cycle pattern of the pathogenic basidiomycetous yeast Cryptococcus neoformans differs from that of the ascomycetous budding yeast Saccharomyces cerevisiae. To clarify the cell cycle control mechanisms at the molecular level, homologues of cell cycle control genes in C. neoformans were cloned and analyzed. Here, we report on the cloning and characterization of genes coding for CDK1 cyclin homologues, in particular, the C. neoformans G1 cyclin. We have identified three putative CDK1 cyclin homologues and two putative CDK5 (PHO85) cyclin homologues from the genome. Complementation tests in an S. cerevisiae G1 cyclin triple mutant confirmed that C. neoformans CLN1 is able to complement S. cerevisiae G1 cyclin deficiency, demonstrating that it is a G1 cyclin homologue. Interestingly, cells deleted of the single Cdk1-G1 cyclin were viable, demonstrating that this gene is not essential. However, it exhibited aberrant budding and cell division and a clear delay in the initiation of DNA synthesis as well as an extensive delay in budding. The fact that the mutant managed to traverse the G1 to M phase may be due to the activities of Pho85-related G1 cyclins. Also, that C. neoformans had only a single Cdk1-G1 cyclin highlighted the importance of keeping in order the commitment to the initiation of DNA synthesis first and then that of budding, as discussed.