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. 2021 Aug 5;2021:10.17912/micropub.biology.000440. doi: 10.17912/micropub.biology.000440

Sorbitol and PKC1 overexpression alleviate temperature sensitivity in chaperonin mutants of Saccharomyces cerevisiae

Aswathy Narayanan 1,2, M Anaul Kabir 1,§
Reviewed by: Anonymous
PMCID: PMC8343407  PMID: 34377964

Abstract

CCT (Chaperonin containing TCP-1) is a constitutively expressed eukaryotic chaperonin complex involved in the proper folding of proteins like actin and tubulin. Temperature sensitive mutants of CCT complex have been employed in various genetic screens, acting as models to study human CCT, the defects of which are implicated in disease conditions like neurodegeneration. Mutants of CCT complex are sensitive to cell wall stress agents. In this study, we have tested the effects of sorbitol and protein kinase C overexpression on the temperature sensitivity of cct mutants. We report that both the factors alleviated temperature sensitivity of cct mutants, indicating the possible role of CCT in maintaining cell wall integrity in S. cerevisiae.

Figure 1. Suppressors of temperature sensitivity in CCT complex chaperonin mutants.

Figure 1. Suppressors of temperature sensitivity in CCT complex chaperonin mutants

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A) Effect of osmotic stabilizer on the temperature sensitivity of cct mutants. Strains B-8728 (WT), DUY326 (cct1-2) and DDY229 (cct4-1) spotted on YPD and incubated at (a) 30°C (b) 35°C and (c) 37°C. Strains B-8728 (WT), DUY326 (cct1-2) and DDY229 (cct4-1) spotted on YPD+1M Sorbitol and incubated at (d) 30°C (e) 35°C and (f) 37°C.

B) Effect of PKC1 and HSP82 overexpression on temperature sensitivity of cct1-2. The mutant cct1-2 transformed with pRSII326 (empty vector), PKC1, HSP82, and CCT1 spotted on uracil-omission media after serial dilution and incubated at (a) 30°C and (b) 37°C.

Description

TRiC (TCP-1 Ring Complex) or CCT (Chaperonin Containing TCP-1) is a multi-subunit, double-ringed, ATP-driven cytosolic eukaryotic chaperonin complex that is constitutively expressed and facilitates the folding of several proteins like actin and tubulin (Frydman et al. 1992; Llorca et al. 1999; Berger et al. 2018). Its role in many cellular processes like differentiation, viral replication, and heart contractility were elucidated recently (Melkani et al. 2017; Knowlton et al. 2018; Ohhara et al. 2019). Its functional defects are also associated with neurodegenerative diseases and cancer (Tam et al. 2006; Boudiaf-Benmammar et al. 2013). Temperature sensitive mutants of Saccharomyces cerevisiae CCT have been used in different studies, especially those involving genetic screens to identify CCT-interacting proteins (Kabir and Sherman 2008). The CCT1–CCT8 genes encode eight different subunits of the complex; the mutant allele cct1-2 has G423D replacement in the conserved ATP binding/hydrolysis domain in the subunit Cct1p. cct4-1 (G345D), the mutation in subunit Cct4p, affects ATP binding, intra and inter-ring co-operativity (Shimon et al. 2008). cct1-2 and cct4-1 grew well at 30°C (permissive temperature), showed retarded growth at 35°C (semi-permissive temperature) and failed to grow at 37°C (restrictive temperature).

Their sensitivity to cell wall antagonists like SDS and congo red are suggestive of cell wall defects in cct mutants (Narayanan et al. 2016). Are the temperature sensitivity and cell wall defects linked in cct mutants? To answer this, we studied the effects of two different suppressors of cell wall defects on the temperature sensitivity of cct mutants.

Osmotic stabilisers like sorbitol are known to rescue cell-wall integrity mutants by preventing cell lysis (Ribas et al. 1991). We observed that the growth of cct1-2 and cct4-1 mutants was restored in presence of 1M sorbitol both at the semi-permissive and restrictive temperatures (Fig. 1A). Mutants in MAPK-activation pathway undergo cell lysis at elevated temperatures, due to the unrepaired cell wall defects (Kamada et al. 1995). The osmoremedial nature of temperature sensitivity in the cct mutants prompted us to study the role of cell wall biogenesis in temperature sensitivity. Towards this end, we overexpressed the PKC1 gene in cct1-2. The PKC1 gene in S. cerevisiae encodes protein kinase C, homolog of human protein kinase C, an important enzyme in the maintenance of cell wall integrity (Levin 2005). Overexpression of PKC1 resulted in the rescue of the temperature-sensitive phenotype of cct mutants at 35°C and 37°C (Fig. 1B).

Wild type copy of the CCT1 gene was also overexpressed that served as the positive control; the overexpression enabled growth at 35°C and 37°C, confirming that the defects observed in the mutant were indeed due to compromised CCT function. HSP82, a yeast HSP90 homolog, was overexpressed in cct1-2 to check if more copies of another chaperone can reverse the defects in the mutant. But, unlike PKC1, HSP82 on overexpression failed to make an impact on the temperature sensitivity of cct mutants. Similar observations were made for HSP40 and HSP90– overexpression of PKC1 was found to ameliorate the temperature sensitivity in these heat shock protein mutants (Wright et al. 2007). Our results suggest the role of CCT, a constitutively expressed cytosolic chaperonin, in maintaining cell wall integrity in S. cerevisiae.

Methods

Yeast extract (1%)- Peptone (2%)- Dextrose (2%) (YPD) medium was used to grow the yeast strains.The cct1-2 and cct4-1 strains were grown at different temperatures in media with and without sorbitol to study the effect of osmotic stabilizers. DUY326 was transformed with pFR22 (YEpU-PKC1) and pCMS154-HSP82 for the overexpression of PKC1 and HSP82, respectively (Wright et al. 2007) using the lithium acetate/PEG method. The transformants were selected on uracil-omission medium. The plates were incubated at 30 °C for two days and the transformants were subcultured in uracil-omission medium. Approximately equal number of cells of the transformants were serially diluted, spotted on YPD and incubated at different temperatures to study the phenotype.

Reagents

Plasmid Description Reference/Source
pRSII326 2- micron based multicopy plasmid with URA3 Chee et al. 2012
YEpU-PKC1 PKC1 cloned in YEp352 Wright et al. 2007
pCMS154-HSP82 HSP82 cloned in pRS426 Wright et al. 2007
pAB2477 CCT1 URA3 CEN/ARS
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Strain Genotype Figure Source
B-8728 MATa ura3-52 trp1-Δ63 leu2-Δ1 GAL2 Figure 1A Lin et al. 1997
DUY326 MATa ura3-52 lys2-801 ade2-201 cct1-1 leu2-3,-112 Figure 1A T C Huffaker
DDY229 MATa cct4-1 leu2-3,112 lys2-801 ura3-52 Figure 1A D G Drubin
D094 DUY326 transformed with pAB2477 Figure 1B This study
D352 DUY326 transformed with pRS11326 Figure 1B This study
D353 DUY326 transformed with YEpU-PKC1 Figure 1B This study
D354 DUY326 transformed with pCMS154-HSP82 Figure 1B This study
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Acknowledgments

Acknowledgments

We are grateful to D. G. Drubin (University of California, USA), T.C. Huffaker (Cornell University, Ithaca, NY) and M.R. Culbertson (University of Wisconsin, USA) for providing the cct mutant strains. We thank J. L. Brodsky for the plasmids pFR22 and pCMS154-HSP82.

Funding

This work was supported by Council of Scientific and Industrial Research (CSIR), India [Grant No: 37(1571)/12/EMRII].

References

  1. Berger J, Berger S, Li M, Jacoby AS, Arner A, Bavi N, Stewart AG, Currie PD. In Vivo Function of the Chaperonin TRiC in α-Actin Folding during Sarcomere Assembly. Cell Rep. 2018 Jan 01;22(2):313–322. doi: 10.1016/j.celrep.2017.12.069. [DOI] [PubMed] [Google Scholar]
  2. Boudiaf-Benmammar C, Cresteil T, Melki R. The cytosolic chaperonin CCT/TRiC and cancer cell proliferation. PLoS One. 2013 Apr 16;8(4):e60895–e60895. doi: 10.1371/journal.pone.0060895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Frydman J, Nimmesgern E, Erdjument-Bromage H, Wall JS, Tempst P, Hartl FU. Function in protein folding of TRiC, a cytosolic ring complex containing TCP-1 and structurally related subunits. EMBO J. 1992 Dec 01;11(13):4767–4778. doi: 10.1002/j.1460-2075.1992.tb05582.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Kabir MA, Sherman F. Overexpressed ribosomal proteins suppress defective chaperonins in Saccharomyces cerevisiae. FEMS Yeast Res. 2008 Aug 01;8(8):1236–1244. doi: 10.1111/j.1567-1364.2008.00425.x. [DOI] [PubMed] [Google Scholar]
  5. Kamada Y, Jung US, Piotrowski J, Levin DE. The protein kinase C-activated MAP kinase pathway of Saccharomyces cerevisiae mediates a novel aspect of the heat shock response. Genes Dev. 1995 Jul 01;9(13):1559–1571. doi: 10.1101/gad.9.13.1559. [DOI] [PubMed] [Google Scholar]
  6. Knowlton JJ, Fernández de Castro I, Ashbrook AW, Gestaut DR, Zamora PF, Bauer JA, Forrest JC, Frydman J, Risco C, Dermody TS. The TRiC chaperonin controls reovirus replication through outer-capsid folding. Nat Microbiol. 2018 Mar 12;3(4):481–493. doi: 10.1038/s41564-018-0122-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Levin DE. Cell wall integrity signaling in Saccharomyces cerevisiae. Microbiol Mol Biol Rev. 2005 Jun 01;69(2):262–291. doi: 10.1128/MMBR.69.2.262-291.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Lin P, Cardillo TS, Richard LM, Segel GB, Sherman F. Analysis of mutationally altered forms of the Cct6 subunit of the chaperonin from Saccharomyces cerevisiae. Genetics. 1997 Dec 01;147(4):1609–1633. doi: 10.1093/genetics/147.4.1609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Llorca O, Smyth MG, Carrascosa JL, Willison KR, Radermacher M, Steinbacher S, Valpuesta JM. 3D reconstruction of the ATP-bound form of CCT reveals the asymmetric folding conformation of a type II chaperonin. Nat Struct Biol. 1999 Jul 01;6(7):639–642. doi: 10.1038/10689. [DOI] [PubMed] [Google Scholar]
  10. Melkani GC, Bhide S, Han A, Vyas J, Livelo C, Bodmer R, Bernstein SI. TRiC/CCT chaperonins are essential for maintaining myofibril organization, cardiac physiological rhythm, and lifespan. FEBS Lett. 2017 Oct 10;591(21):3447–3458. doi: 10.1002/1873-3468.12860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Narayanan A, Pullepu D, Reddy PK, Uddin W, Kabir MA. Defects in Protein Folding Machinery Affect Cell Wall Integrity and Reduce Ethanol Tolerance in S. cerevisiae. Curr Microbiol. 2016 Mar 18;73(1):38–45. doi: 10.1007/s00284-016-1024-x. [DOI] [PubMed] [Google Scholar]
  12. Ohhara Y, Nakamura A, Kato Y, Yamakawa-Kobayashi K. Chaperonin TRiC/CCT supports mitotic exit and entry into endocycle in Drosophila. PLoS Genet. 2019 Apr 29;15(4):e1008121–e1008121. doi: 10.1371/journal.pgen.1008121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ribas JC, Diaz M, Duran A, Perez P. Isolation and characterization of Schizosaccharomyces pombe mutants defective in cell wall (1-3)beta-D-glucan. J Bacteriol. 1991 Jun 01;173(11):3456–3462. doi: 10.1128/jb.173.11.3456-3462.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Shimon L, Hynes GM, McCormack EA, Willison KR, Horovitz A. ATP-induced allostery in the eukaryotic chaperonin CCT is abolished by the mutation G345D in CCT4 that renders yeast temperature-sensitive for growth. J Mol Biol. 2008 Jan 15;377(2):469–477. doi: 10.1016/j.jmb.2008.01.011. [DOI] [PubMed] [Google Scholar]
  15. Tam S, Geller R, Spiess C, Frydman J. The chaperonin TRiC controls polyglutamine aggregation and toxicity through subunit-specific interactions. Nat Cell Biol. 2006 Sep 17;8(10):1155–1162. doi: 10.1038/ncb1477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Wright CM, Fewell SW, Sullivan ML, Pipas JM, Watkins SC, Brodsky JL. The Hsp40 molecular chaperone Ydj1p, along with the protein kinase C pathway, affects cell-wall integrity in the yeast Saccharomyces cerevisiae. Genetics. 2007 Jan 21;175(4):1649–1664. doi: 10.1534/genetics.106.066274. [DOI] [PMC free article] [PubMed] [Google Scholar]

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