Saccharomyces cerevisiae survival against heat stress entails a communication between CCT and cell wall integrity pathway.

The chaperonin TRiC/CCT is cytosolic cylindrical complex of 16 subunits encoded by eight essential genes CCT1-8. It contributes to folding 10% of cellular polypeptides in yeast. The strain carrying substitution point mutation G412E in the equatorial domain of Cct7p resulted in the improper folding of substrates. In this study, the Cct7p mutant exhibited sensitivity to non-optimal growth temperatures and cell wall stressors. Heat shock is known to disrupt cell wall and protein stability in budding yeast. Mitogen-activated protein kinase-mediated cell wall integrity pathway gets activated to compensate the perturbed cell wall. Overexpression of the PKC1 and SLT2 genes of MAPK signaling pathway in mutant rescued the growth and cell division defects. Additionally, the genes of the CWI pathway such as SED1, GFA1, PIR1, and RIM21 are down-regulated. The Cct7p mutant strain (G412E) is unable to withstand the heat stress due to the underlying defects in protein folding and cell wall maintenance. Taken together, our results strongly indicate the interaction between CCT and cell wall integrity pathway.

CSU52, a novel regulator functions as a repressor of L-sorbose utilization in Candida albicans.

BACKGROUND AND OBJECTIVES: Monosomy of chromosome 5 associated with utilization of non-canonical sugar L-sorbose is one of the well-studied aneuploidies in Candida albicans. Stress-induced ploidy changes are crucial determinants for pathogenicity and genetic diversity in C. albicans. The five scattered regulatory regions (A, B, C, 135, and 139) comprising of two functionally redundant pathways (SUR1 and SUR2) were found to be responsible for the growth on L-sorbose. So far, three genes such as CSU51, CSU53 and CSU57 have been identified in region A, region 135 and region C, respectively. In this study we have verified the role of region B in this regulatory pathway. MATERIALS AND METHODS: We employed a combinatorial gene deletion approach to verify the role of region B followed by co-over expression studies and qRT-PCR to identify the regulatory role of this region. RESULTS: We confirmed the role of region B in the regulation of SOU1 gene expression. The qRT-PCR results showed that regulation occurs at transcriptional level along with other two regions in SUR1 pathway. A previously uncharacterized open reading frame in region B has been implicated in this regulation and designated as CSU52. Integrating multiple copies of CSU52 in the genome at tandem, suppresses the growth of recipient strain on L-sorbose, establishing it as a repressor of SOU1 gene. CONCLUSION: This finding completes the identification of regulators in SUR1 pathway. This result paves the way to study the underlying molecular mechanisms of SOU1 gene regulation that in-turn helps to understand stress induced aneuploidy.

CSU57 encodes a novel repressor of sorbose utilization in opportunistic human fungal pathogen Candida albicans.

Human fungal pathogen Candida albicans cannot utilize L-sorbose as a sole carbon source. However, chromosome 5 monosomic strains can grow on sorbose as repressors present on this chromosome get diminished allowing the expression of sorbose utilization gene (SOU1) located on chromosome 4. Functional identification of these repressors has been a difficult task as they are scattered on a large portion of the right arm of chromosome 5. Herein, we have applied the telomere-mediated chromosomal truncation approach to identify a novel repressor for sorbose utilization in this pathogen. Multiple systematic chromosomal truncations were performed on the right arm of Chr5 in the background of csu51∆/CSU51 to minimize the functional region to 6-kb chromosomal stretch. Further, truncation that removes the part of Orf19.3942 strongly suggested its role in sorbose utilization. However, compelling evidence comes from the observation that truncation at 1,044.288-kb position of Chr5 in the strain csu51∆/CSU51 orf19.3942∆/Orf.19.3942 produced Sou+ phenotype; otherwise, the strain remains Sou- . This confirms beyond doubt the role of Orf.19.3942 in the regulation of sorbose utilization and designated as CSU57. Comparison of SOU1 gene expression of Sou+ strains with wild type suggested its role at transcriptional level. Strain carrying double disruption of CSU57 remains Sou- . Co-overexpression of SOU1 and CSU57 together does not make the recipient strain Sou- ; however, multiple tandem copies of CSU57 produced diminished growth compared with control suggesting that it is a weak repressor. Taken together, we report that CSU57 encodes a novel repressor of L-sorbose utilization in this pathogen. TAKE AWAY: CSU57 encodes a repressor for L-sorbose utilization in Candida albicans. Csu57p acts in combination with Csu51p and other regulators. Csu57p exerts its repressing effect at transcriptional level of SOU1 gene. Utilization of sorbose positively correlates to the expression of SOU1 gene. Multiple copies of CSU57 can partially suppress Sou+ phenotype.

The interactome of CCT complex - A computational analysis.

The eukaryotic chaperonin, CCT (Chaperonin Containing TCP1 or TriC-TCP-1 Ring Complex) has been subjected to physical and genetic analyses in S. cerevisiae which can be extrapolated to human CCT (hCCT), owing to its structural and functional similarities with yeast CCT (yCCT). Studies on hCCT and its interactome acquire an additional dimension, as it has been implicated in several disease conditions like neurodegeneration and cancer. We attempt to study its stress response role in general, which will be reflected in the aspects of human diseases and yeast physiology, through computational analysis of the interactome. Towards consolidating and analysing the interactome data, we prepared and compared the unique CCT-interacting protein lists for S. cerevisiae and H. sapiens, performed GO term classification and enrichment studies which provide information on the diversity in CCT interactome, in terms of protein classes in the data set. Enrichment with disease-associated proteins and pathways highlight the medical importance of CCT. Different analyses converge, suggesting the significance of WD-repeat proteins, protein kinases and cytoskeletal proteins in the interactome. The prevalence of proteasomal subunits and ribosomal proteins suggest a possible cross-talk between protein-synthesis, folding and degradation machinery. A network of chaperones and chaperonins that function in combination can also be envisaged from the CCT interactome-Hsp70 interactome analysis.

Defects in Protein Folding Machinery Affect Cell Wall Integrity and Reduce Ethanol Tolerance in S. cerevisiae.

The chaperonin complex CCT/TRiC (chaperonin containing TCP-1/TCP-1 ring complex) participates in the folding of many crucial proteins including actin and tubulin in eukaryotes. Mutations in genes encoding its subunits can affect protein folding and in turn, the physiology of the organism. Stress response in Saccharomyces cerevisiae is important in fermentation reactions and operates through overexpression and underexpression of genes, thus altering the protein profile. Defective protein folding machinery can disturb this process. In this study, the response of cct mutants to stress conditions in general and ethanol in specific was investigated. CCT1 mutants showed decreased resistance to different conditions tested including osmotic stress, metal ions, surfactants, reducing and oxidising agents. Cct1-3 mutant with the mutation in the conserved ATP-binding region showed irreversible defects than other mutants. These mutants were found to have inherent cell wall defects and showed decreased ethanol tolerance. This study reveals that cell wall defects and ethanol sensitivity are linked. Genetic and proteomic analyses showed that the yeast genes RPS6A (ribosomal protein), SCL1 (proteasomal subunit) and TDH3 (glyceraldehyde-3-phosphate dehydrogenase) on overexpression, improved the growth of cct1-3 mutant on ethanol. We propose the breakdown of common stress response pathways caused by mutations in CCT complex and the resulting scarcity of functional stress-responsive proteins, affecting the cell's defence against different stress agents in cct mutants. Defective cytoskeleton and perturbed cell wall integrity reduce the ethanol tolerance in the mutants which are rescued by the extragenic suppressors.