As polypeptide chains emerge from the ribosome, they interact with several chaperone machines. Among these are components of the RAC complex, which includes ribosome-binding Hsp70 and Hsp40 chaperones that promote efficient polypeptide translation (50
). Other members of the Hsp70 and Hsp110 families also participate in nascent chain binding (55
). More specific chaperones interact with nascent chains from distinct protein families, such as Cdc37, which protects nascent protein kinases from rapid degradation by the proteasome. In the present study, we characterized the type I Hsp40, Ydj1, as also having a protective role in protein kinase biogenesis at the earliest stages posttranslation. Ydj1 also has a role in kinase biogenesis that is distinct from Cdc37, by controlling the rate of kinase maturation as well as the yield of mature kinase.
Ydj1's ability to protect nascent protein kinases results from a direct effect and an indirect one via Hsp70. Of the two, it is the latter that has the greatest contribution to stabilizing protein kinases. In the cdc37S14A
mutant, overexpression of wild-type YDJ1
or the mutant ydj1H34Q
led to a temporary stabilization of Cdc28. Since the ydj1H34Q
mutant cannot interact with Hsp70, these findings suggest that the intrinsic chaperone function of Ydj1 promoted the stabilization, albeit weakly. Furthermore, the overall effects of expressing ydj1H34Q
in place of wild-type Ydj1 were negligible at steady state for Tpk2 (Fig. ), suggesting that Hsp70 binding is required for complete protection against degradation. As noted previously, Ste11 kinase synthesized in a ydj1
Δ mutant had decreased amounts of bound Hsp70 (32
), reinforcing the notion that Ydj1 interacts with nascent kinases in order to protect them, but this effect must be integrated with subsequent binding to Hsp70 to avoid degradation. Notably, Hsp90 and Cdc37 could still bind to Ste11 even in a ydj1
Δ strain (32
). Such binding likely represents a quality control step in kinase maturation that is at least partially distinct from that managed by Ydj1. This is because even in the absence of Ydj1 there is a strong effect of GA in promoting kinase degradation (Fig. ). These data therefore point to a role for both Hsp70 and Hsp90 chaperone machines in protecting newly synthesized kinases from degradation, as well as helping them to fold. Interestingly, both chaperones are upregulated in cells with YDJ1
deleted (Fig. ).
The notion that both Hsp70 and Hsp90 contribute to kinase stability fits with our finding that Tpk2 degradation is biphasic. The first phase is during the 10-min pulse-labeling period, in a manner similar to the way in which Tpk2 is degraded in the cdc37
). Like Cdc37, therefore, Ydj1 protects nascent chains and, in its absence, the newly made kinase is largely directed toward the ubiquitin/proteasome system. Tpk2 that escapes initial triage either folds slowly or is subsequently degraded in a slower process. We did not observe the fast degradation of Cdc28 in a ydj1
Δ strain, a finding consistent with a similar finding for the cdc37
mutant (Fig. ) (36
). Instead, there is a delay in Cdc28 maturation, followed by a slow decrease in kinase levels. For both Cdc28 and Tpk2, however, the rate of kinase degradation is much more pronounced in the cdc37
mutant than in the ydj1
Δ strain. Despite this difference, Ydj1 and Cdc37 both contribute in a similar manner to the folding process; each stabilizes nascent kinases and helps promote binding to another chaperone, Hsp70 in the case of Ydj1 and Hsp90 in the case of Cdc37. Although Ydj1 is an abundant protein, it is still limiting in its capacity to protect newly synthesized kinases. When overexpressed, Ydj1 promotes greater stability of Tpk2 and Slt2 kinases in the presence of GA. Similarly, overexpressed Ydj1 or ydj1H34Q
delayed the degradation of unstable Cdc28 in cdc37S14A
mutant cells (Fig. and Fig. ).
Although our results provide strong support for a protective role for Ydj1 in kinase biogenesis, this is not a universal function. There have been several published reports showing that there is reduced degradation of unstable proteins in a mutant of YDJ1
). However, we found that the expression of ydj1-151
largely suppressed the effect of complete YDJ1
deletion and led to the accumulation of folded Tpk2 as measured by enzyme activity (data not shown). Our model, therefore, is that Ydj1 functions to promote kinase folding but is not involved in targeting misfolded kinases to the degradation machinery, at least under nonstress conditions.
Besides acting to protect nascent kinases, Ydj1 also affects the rate at which maturation takes place. This effect occurred in ydj1Δ and ydj1H34Q mutants (Fig. and data not shown), indicating that the effect depends more on the relationship between Ydj1 and Hsp70 than on the intrinsic chaperone action of Ydj1 itself. The basis for the reduced rate of kinase maturation in the ydj1 mutations, however, remains obscure. One possibility relates to the finding that ydj1 mutants grow slowly and thus have reduced rates of metabolic processes. However, pulse-chase analyses of Tpk2 maturation in other chaperone mutants that grow slowly (cpr7Δ and sse1Δ) suggested that this is not the case (not shown).
The ability of Ydj1 to protect nascent kinases has some specificity since SIS1
overexpression was unable to suppress defects in kinase biogenesis in a ydj1
Δ mutant. The lack of suppression occurs despite the finding that SIS1
overexpression can improve growth of ydj1
Δ cells (8
). Furthermore, the growth of ydj1
Δ cells can be improved by just increasing the abundance of the J domain, suggesting that impairment to Hsp70's functional cycle is the main factor that limits growth of ydj1
Δ cells (45
). Tests of which domains impart the functional specificity were largely negative but demonstrated that the chaperone modules specific to type I and type II Hsp40s were not responsible (Fig. ). Further support for this view derives from the finding that mammalian Hdj2 (type I) and Hsp40 (type I) are interchangeable for their function in folding of progesterone receptor and Chk1 protein kinase (13
). Based on this, the specificity with which we observed Ydj1 function may have more to do with how Sis1 is unable to interact with kinases or nuclear receptors in the presence of Hsp70. Previous studies showed that Ydj1 is capable of refolding luciferase in association with Hsp70 in vitro, whereas Sis1 does not function as efficiently in this capacity (34
). The basis for their differential function is due in part to each having a distinct chaperone module, which can specify the function of Sis1 or Ydj1 in chimeric molecules (19
). We note, however, that Sis1 also has a more stable interaction with the Hsp70 C terminus than does Ydj1 (33
). The C-terminal region of Hsp70 interacts with the peptide-binding groove in the chaperone module of Sis1, suggesting that there could be competitive interactions. This might preclude Sis1 binding to nascent kinases in the presence of Hsp70. We speculate that such binding of Hsp70 to the Sis1 chaperone module is disrupted in the YSY chimera and that this is why it proves so effective compared to Sis1 alone (Fig. ).
In conclusion, our combined findings suggest a model in which Ydj1 has two specific functions in protein kinase folding. The first involves protecting the newly synthesized kinase chain from degradation, and the second involves promoting efficient folding and maturation. Ydj1 by itself can protect nascent kinases, but not to the same extent that it can in association with Hsp70. The unexpected finding that Ydj1 can affect the rate of kinase maturation presents a new paradigm for its action in cellular quality control.