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Kayla McKaveney, Peter Kuhn, Elizabeth A. Craig Department of Biochemistry April 23, 2013

Saccharomyces cerevisiae chaperones Ssb and Zuo1 influence long-term cell viabilit y and population growth. Kayla McKaveney, Peter Kuhn, Elizabeth A. Craig Department of Biochemistry April 23, 2013. Outline. Molecular chaperones: background in brief Ssb and Zuo1 in yeast

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Kayla McKaveney, Peter Kuhn, Elizabeth A. Craig Department of Biochemistry April 23, 2013

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  1. Saccharomyces cerevisiaechaperones Ssb and Zuo1 influence long-term cell viability and population growth Kayla McKaveney, Peter Kuhn, Elizabeth A. Craig Department of Biochemistry April 23, 2013

  2. Outline • Molecular chaperones: background in brief • Ssb and Zuo1 in yeast • A model for two classes of Ssb and Zuo1 phenotypes • Future direction • SsbATPase assay • Cell-based assays

  3. Molecular Chaperones • Assist in protein folding • Refoldmis-folded or aggregated proteins • Transport proteins between cellular compartments • Assist in degradation Cartoon courtesy of Lindsay Hoover

  4. Heat Shock 70kDa Protein (Hsp70)

  5. J-protein partner of Hsp70 drives chaperone activity • Stimulates Hsp70 ATPase domain • J-protein required for efficient ATP hydrolysis by the Hsp70 Kampinga and Craig 2010

  6. Zuo1 mutation in a critical amino acid Normal Zuo1 HPD 1 128 433 Zuo1 mutation (H128R) RPD 1 128 433 Zuo1 mutation (H128Q) QPD 1 128 433 Amino acid position

  7. Ssb (Hsp70) and Zuo1 (J-protein) chaperone complex on the ribosome • Zuo1 acts as the ‘switch’ and stimulates Ssb • Ssb has two parts • ATP hydrolysis powers ATPase domain • Hinge in peptide binding domain aids folding Ribosome 40S mRNA Ssz1 60S Zuo1 Ssb polypeptide

  8. Aims • To what extent is ATPase stimulation hindered by the Zuo1 H128R mutation? • In vitro ATPase assay • Are yeast cell density and longevity phenotypes related to the chaperone activity of Zuo1 and Ssb? • In vivo growth assays and programmed cell death (PCD) assessment

  9. Methods Transform DNA into yeast cells Mutate Zuo1 gene using PCR Grow yeast, harvest, and lyse cells Perform cell longevity assays in yeast Extract and purify protein Measure ATPase stimulation of Ssb by Zuo1 variants Investigate involvement of programmed cell death (PCD)

  10. Classic Zuo1-Ssb disruption phenotypes • Cold, translational stress, osmotic stress • HPD is important

  11. Emerging category of Zuo1-Ssb disruption phenotypes • High density culture growth, growth arrest in low nutrient conditions, longevity • Greater dependency on HPD

  12. Zuo1-Ssb disruption prevents growth arrest on nutrient limited media (NLM) Wild-type Δzuo1 • Δssb1/2 NLM Control Zuo1 Δzuo1 • H128R

  13. Ssb deletion mutant outgrows wild-type yeast wild-type Δssb von Plehwe 2009 von Plehwe 2009 Prunuske 2012

  14. Zuo1 H128R results in increased culture densities and chronological life span (CLS) compared to wild-type Zuo1 Peter Kuhn, unpublished data

  15. Enzyme Assay Ssb + labeled ATP Zuo1 (normal or mutant) Zuo1 + Ssb + 1) labeled ADP 2) labeled ATP Huang, et al., 2005.

  16. Programmed Cell Death (PCD) Assays • BrDU staining of dividing cells • Determine cause of increased cell density • DAPI staining for chromatin fragmentation • TUNEL staining for DNA strand breaks Young cells Aged cells Frohlich and Madeo 2001

  17. Conclusions • Zuo1 and Ssb disruption cause two classes of phenotypes • Yeast with Zuo1 H128Q and Zuo1 H128R mutations fail to respond to low nutrient stress Future Direction • Determine degree of Ssb stimulation from Zuo1 variants • Investigate the role of chaperones in nutrient signaling

  18. Acknowledgements • Elizabeth Craig • Peter Kuhn • Tom Ziegelhoffer • Craig Lab

  19. Questions?

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