Ydj1 protects nascent protein kinases from degradation and controls the rate of their maturation

Abstract

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 ydj1Delta mutant, while the remainder was capable of maturing but with reduced kinetics compared to the wild type. Cdc28 maturation was also delayed in the ydj1Delta 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 ydj1Delta 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.

FIG. 1.

Effect of YDJ1 deletion on protein kinase levels. (A) Steady-state level of Tpk2 in wild-type and ydj1Δ yeast cells; (B) Rim11 in wild-type and ydj1Δ cells. Wild-type and ydj1Δ yeast cells were grown overnight at 30°C and kinases identified with anti-TAP. Western blots of the same samples were performed with anti-Pgk1, anti-Ydj1, anti-Hsp70, and anti-Hsp90. Pgk1 levels are shown as a loading control. (C) Steady-state levels of kinases in ydj1Δ strain. The results are expressed as the percentages of levels in wild-type cells in each case based on Western blot analysis. The bars indicate the standard deviation from three to four independent experiments. WT, wild type.

FIG. 2.

Solubility and activity of protein kinases in ydj1Δ mutant strains. (A) Solubility assay of Tpk2 kinase. The solubility of Tpk2 was assayed in wild-type and ydj1Δ cells by comparing equal amounts of total lysate (T), supernatant (S), and pellet (P) fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The same blot was analyzed with anti-Hsp70, anti-Pgk1, and anti-Atp2, which encodes the β subunit of the mitochondrial F1 ATPase. (B) Tpk2 activity in wild-type and ydj1Δ mutant strains. The upper panel shows the Tpk2 activity after immunoprecipitation of the kinase from various amounts of wild-type extract (expressed in milligrams) and from a 3-mg portion of extracts from ydj1Δ cells. The panel shows the fluorescence from a labeled peptide whose migration in an agarose gel depends on phosphorylation by immunoprecipitated Tpk2. The lower panel shows the amount of Tpk2 in each reaction as determined by Western blotting. The bar graph at the right shows the quantitation of Tpk2 activity after normalization to the amount of enzyme in the assay. Bars indicate the standard error of the mean values of three independent experiments. (C) Assay of Rim11 activity. The upper panel shows Western blot results of TAP-Rim11 immunoprecipitates and the amount of ³²P-labeled myelin basic protein (MBP) used in the assay. The panel on the right shows the MBP data quantified. Bars represent the standard error (n = 3). WT, wild type.

FIG. 3.

Pulse-chase analysis of Tpk2 kinase in the wild type and the ydj1Δ mutant. (A) Pulse-chase analysis of TAP-tagged Tpk2 in wild-type (YDJ1) and ydj1Δ mutant cells. The chase times are indicated in minutes. (B) Pulse-chase analysis of wild-type cells after a 2-min pulse and chase from 0 to 10 min. (C) Pulse-chase analysis of TAP-Tpk2 in ydj1Δ cells. Cells were treated with dimethyl sulfoxide or 100 μM MG132 for 30 min before labeling. The strain background is erg6Δ, which improves MG132 permeability. The chase times are indicated in minutes. Mature (M, phosphorylated) and immature (I, nonphosphorylated) forms of Tpk2 are denoted by arrows. The relative amounts of mature and immature kinase in each lane were quantified, and the numbers appear below the panel.

FIG. 4.

Pulse-chase analysis of Cdc28. (A) Pulse-chase analysis of untagged Cdc28 immunoprecipitated with anti-PSTAIRE (which recognizes a peptide in the α-C helix) in wild-type and ydj1Δ mutant cells. Chase times are indicated in minutes. Cdc28 is denoted by arrow. The band labeled with an asterisk is nonspecific. The inset shows the difference in mobility of Cdc28 at 0 and 30 min of chase time. I, immature form of Cdc28; M, mature form. (B) Quantitation of Cdc28 levels in wild-type (WT) and ydj1Δ cells after phosphorimaging. The results are expressed as the percentages of the levels in wild-type cells at 0-min chase. Bars indicate the standard deviations of three independent experiments. Bars: ▪, wild type; , ydj1Δ mutant. (C) Pulse-chase analysis of TAP-tagged Cdc28 in wild-type and ydj1Δ cells as described above.

FIG. 5.

Effect of Ydj1 on the stability of nascent protein kinases under conditions that promote their degradation. (A) Effect of GA treatment on the stability of TAP-Slt2 kinase in wild-type and ydj1Δ cells, as indicated. Chase times are given in hours after pulse-labeling in the absence and presence of 50 μM GA. (B) Quantitation of Slt2 based on the data shown in panel A and other data (n = 3 ± the standard error). (C) Pulse-chase analysis of TAP-Tpk2 in the absence and presence of 50 μM GA in ydj1Δ cells. The chase times are indicated in hours. Mature (M) and immature (I) forms of Tpk2 are indicated. (D) Pulse-chase analysis of TAP-Tpk2 from cells containing pGAL-YDJ1 under noninducing condition (glucose; Glu) and inducing conditions (galactose, Gal). The lower panel shows pulse-chase results for Tpk2 in cells containing galactose but not the pGAL-YDJ1 plasmid. (E) Western blot analysis of Ydj1 protein before and after overexpression using the same cultures as in panel D. Pgk1 is shown as a loading control. (F) Effect of YDJ1 overexpression on TAP-Slt2 stability in the presence of GA (50 μM). Inducing and noninduced states are as described in panel D.

FIG. 6.

Ydj1 protects against degradation of unstable Cdc28 in a cdc37 mutant strain. (A) Pulse-chase analysis of Cdc28 in wild-type and cdc37^S14A mutant cells without or with overexpression of YDJ1 or the mutant ydj1^H34Q. Chase times are given in minutes. Immature (I) and mature (M) forms of Cdc28 are indicated. The band labeled with an asterisk is nonspecific. (B) Quantitation of the data shown in panel A. Symbols: •, wild type; ▪, cdc37^S14A mutant; ▴, cdc37^S14A mutant with YDJ1 overexpressed; ○, cdc37^S14A mutant with ydj1^H34Q overexpressed. (C) Western blot analysis of Cdc28 in wild-type and cdc37^S14A mutant cells (S14A) either with (+) or without (−) YDJ1 overexpression. The lower panel shows the Ydj1 levels. The band labeled with an asterisk is nonspecific. WT, wild type.

FIG. 7.

Steady-state TAP-Tpk2 levels in ydj1 mutants. (A) Western blot analysis of TAP-Tpk2 (Tpk2) in wild-type and ydj1Δ cells without or with the ydj1^H34Q mutant expressed from a low-copy-number plasmid. The middle panel shows Ydj1 levels, and the lower panel shows the levels of Pgk1 as a loading control. (B) Western blot analysis of wild-type and ydj1Δ mutants without or with overexpression of SIS1. The middle panel shows the levels of Pgk1 as a loading control, while the lower panel shows the levels of Ydj1 and Sis1. (C) Western blot of TAP-Tpk2 in wild-type and ydj1Δ cells with or without Sis1, YSY, and SYS chimeric chaperones as indicated. The middle panel shows Pgk1, and the lower panel shows the levels of Sis1 and Ydj1/Sis1 chimeras using anti-Sis1. (D) Effect of Ydj1's G/F region in kinase accumulation. Tpk2 levels were analyzed in wild-type and Ydj1ΔG/F-expressing cells in a ydj1Δ background. (E) Effect of bacterial DnaJ in kinase accumulation. The Tpk2 levels were measured after overexpression of DnaJ in wild-type and ydj1Δ yeast strains. DnaJ expression was carried out after induction with galactose for 8 h. Tpk2 was identified with anti-TAP antibody. The kinase levels were quantified and are shown as a percentage of the wild-type levels. Western blot anlayses with the same samples were performed with anti-Pgk1, anti-DnaJ, and anti-Ydj1. Pgk1 levels served as a loading control. WT, wild type.