Equivalent Mutations in the Eight Subunits of the Chaperonin CCT Produce Dramatically Different Cellular and Gene Expression Phenotypes |
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Authors: | Maya Amit Michal Nadler-Holly Ester Feldmesser Keith R. Willison |
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Affiliation: | 1 Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel 2 Section of Cell and Molecular Biology, Institute of Cancer Research, Chester Beatty Laboratories, London SW3 6JB, UK 3 Department of Biological Services, Weizmann Institute of Science, Rehovot 76100, Israel 4 Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel |
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Abstract: | The eukaryotic cytoplasmic chaperonin-containing TCP-1 (CCT) is a complex formed by two back-to-back stacked hetero-octameric rings that assists the folding of actins, tubulins, and other proteins in an ATP-dependent manner. Here, we tested the significance of the hetero-oligomeric nature of CCT in its function by introducing, in each of the eight subunits in turn, an identical mutation at a position that is conserved in all the subunits and is involved in ATP hydrolysis, in order to establish the extent of ‘individuality’ of the various subunits. Our results show that these identical mutations lead to dramatically different phenotypes. For example, Saccharomyces cerevisiae yeast cells with the mutation in subunit CCT2 display heat sensitivity and cold sensitivity for growth, have an excess of actin patches, and are the only strain here generated that is pseudo-diploid. By contrast, cells with the mutation in subunit CCT7 are the only ones to accumulate juxtanuclear protein aggregates that may reflect an impaired stress response in this strain. System-level analysis of the strains using RNA microarrays reveals connections between CCT and several cellular networks, including ribosome biogenesis and TOR2, that help to explain the phenotypic variability observed. |
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Keywords: | CCT, chaperonin-containing TCP-1 FACS, fluorescence-activated cell sorting GFP, green fluorescent protein Ubc, ubiquitin conjugating VHL, von Hippel-Lindau FDR, false discovery rate CBP, calmodulin-binding peptide UTR, untranslated region PBS, phosphate-buffered saline |
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