Ydj1 Protects Nascent Protein Kinases from Degradation and Controls the Rate of Their Maturation

Department of Biology, City College New York, Convent Avenue at 138th Street, New York, NY 10031, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 08/2008; 28(13):4434-44. DOI: 10.1128/MCB.00543-08
Source: PubMed


Ydj1 is a Saccharomyces cerevisiae Hsp40 molecular chaperone that functions with Hsp70 to promote polypeptide folding. We identified Ydj1 as being important
for maintaining steady-state levels of protein kinases after screening several chaperones and cochaperones in gene deletion
mutant strains. Pulse-chase analyses revealed that a portion of Tpk2 kinase was degraded shortly after synthesis in a ydj1Δ mutant, while the remainder was capable of maturing but with reduced kinetics compared to the wild type. Cdc28 maturation
was also delayed in the ydj1Δ mutant strain. Ydj1 protects nascent kinases in different contexts, such as when Hsp90 is inhibited with geldanamycin or
when CDC37 is mutated. The protective function of Ydj1 is due partly to its intrinsic chaperone function, but this is minor compared
to the protective effect resulting from its interaction with Hsp70. SIS1, a type II Hsp40, was unable to suppress defects in kinase accumulation in the ydj1Δ mutant, suggesting some specificity in Ydj1 chaperone action. However, analysis of chimeric proteins that contained the
chaperone modules of Ydj1 or Sis1 indicated that Ydj1 promotes kinase accumulation independently of its client-binding specificity.
Our results suggest that Ydj1 can both protect nascent chains against degradation and control the rate of kinase maturation.

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Available from: Avrom J Caplan, Oct 10, 2015
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    • "Although loss of Ydj1 results in decreased Cdk1-mediated phosphorylation of the Cln3 PEST domain and stabilization of Cln3 (Yaglom et al., 1996), ydj1D cells are shifted toward G1, a defect suppressed through overexpression of Cln3 (Vergé s et al., 2007). The decreased Cln3 phosphorylation and lower Ssa1 T36 phosphorylation in cells lacking Ydj1 may each be explained by a decrease in Cdk1 (and perhaps Pho85) abundance in ydj1D cells (Mandal et al., 2008). Nonetheless, in cells lacking Ydj1, the Ssa1-Cln3 interaction becomes constitutive, perhaps limiting Cln3 access to the nucleus, and thereby partially uncoupling PEST domain phosphorylation, Cln3 stability, and Cdk1-Cln3 activity. "
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    ABSTRACT: In budding yeast, the essential functions of Hsp70 chaperones Ssa1-4 are regulated through expression level, isoform specificity, and cochaperone activity. Suggesting a novel regulatory paradigm, we find that phosphorylation of Ssa1 T36 within a cyclin-dependent kinase (CDK) consensus site conserved among Hsp70 proteins alters cochaperone and client interactions. T36 phosphorylation triggers displacement of Ydj1, allowing Ssa1 to bind the G1 cyclin Cln3 and promote its degradation. The stress CDK Pho85 phosphorylates T36 upon nitrogen starvation or pheromone stimulation, destabilizing Cln3 to delay onset of S phase. In turn, the mitotic CDK Cdk1 phosphorylates T36 to block Cln3 accumulation in G2/M. Suggesting broad conservation from yeast to human, CDK-dependent phosphorylation of Hsc70 T38 similarly regulates Cyclin D1 binding and stability. These results establish an active role for Hsp70 chaperones as signal transducers mediating growth control of G1 cyclin abundance and activity.
    Cell 12/2012; 151(6):1308-18. DOI:10.1016/j.cell.2012.10.051 · 32.24 Impact Factor
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    • "The main difference is that we observed a role for Ubr2 in cytosolic quality control processes whereas they did not. Furthermore, they described how Ydj1 promotes degradation while we showed previously that Ydj1 protects newly synthesized protein kinases from that fate (Mandal et al., 2008). One clear difference between our studies and those of Heck et al. (2010) is that we studied predominantly wildtype proteins (e.g., Tpk2) whose folding pathways are blocked (with the exception of the AZC experiment shown in Figure 5), while they studied mutant proteins or proteins mislocalized from other compartments. "
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    ABSTRACT: Quality control systems facilitate polypeptide folding and degradation to maintain protein homeostasis. Molecular chaperones promote folding, whereas the ubiquitin/proteasome system mediates degradation. We show here that Saccharomyces cerevisiae Ubr1 and Ubr2 ubiquitin ligases promote degradation of unfolded or misfolded cytosolic polypeptides. Ubr1 also catalyzes ubiquitinylation of denatured but not native luciferase in a purified system. This activity is based on the direct interaction of denatured luciferase with Ubr1, although Hsp70 stimulates polyubiquitinylation of the denatured substrate. We also report that loss of Ubr1 and Ubr2 function suppressed the growth arrest phenotype resulting from chaperone mutation. This correlates with increased protein kinase maturation and indicates partitioning of foldable conformers toward the proteasome. Our findings, based on the efficiency of this quality control system, suggest that the cell trades growth potential to avert the potential toxicity associated with accumulation of unfolded or misfolded proteins. Ubr1 and Ubr2 therefore represent E3 components of a novel quality control pathway for proteins synthesized on cytosolic ribosomes.
    Molecular biology of the cell 05/2010; 21(13):2102-16. DOI:10.1091/mbc.E10-02-0098 · 4.47 Impact Factor
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    • "Interruption of the Hsp70 – Hsp90-folding cycle through genetic or pharmacological means, such as the specific inhibitor geldanamycin (GA), can result in ubiquitinylation and degradation of the client protein (Whitesell and Lindquist, 2005). Identification of the Hsp70 activator Ydj1 as an important regulator of protein kinase stability when forward progress through the Hsp90 system is compromised suggests that Hsp70 is a major nexus of client fate evaluation and that other Hsp70-regulating partners may likewise influence this decision (Mandal et al., 2008). Sse1 has been physically and genetically linked to the Hsp90 chaperone complex in yeast, although its precise mode of action is unclear (Liu et al., 1999; Goeckeler et al., 2002; Lee et al., 2004). "
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    ABSTRACT: Heat shock protein 70 (Hsp70) plays a central role in protein homeostasis and quality control in conjunction with other chaperone machines, including Hsp90. The Hsp110 chaperone Sse1 promotes Hsp90 activity in yeast, and functions as a nucleotide exchange factor (NEF) for cytosolic Hsp70, but the precise roles Sse1 plays in client maturation through the Hsp70-Hsp90 chaperone system are not fully understood. We find that upon pharmacological inhibition of Hsp90, a model protein kinase, Ste11DeltaN, is rapidly degraded, whereas heterologously expressed glucocorticoid receptor (GR) remains stable. Hsp70 binding and nucleotide exchange by Sse1 was required for GR maturation and signaling through endogenous Ste11, as well as to promote Ste11DeltaN degradation. Overexpression of another functional NEF partially compensated for loss of Sse1, whereas the paralog Sse2 fully restored GR maturation and Ste11DeltaN degradation. Sse1 was required for ubiquitinylation of Ste11DeltaN upon Hsp90 inhibition, providing a mechanistic explanation for its role in substrate degradation. Sse1/2 copurified with Hsp70 and other proteins comprising the "early-stage" Hsp90 complex, and was absent from "late-stage" Hsp90 complexes characterized by the presence of Sba1/p23. These findings support a model in which Hsp110 chaperones contribute significantly to the decision made by Hsp70 to fold or degrade a client protein.
    Molecular biology of the cell 03/2010; 21(9):1439-48. DOI:10.1091/mbc.E09-09-0779 · 4.47 Impact Factor
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