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[摘要]：The Saccharomyces cerevisiae Hal3 protein is a moonlighting protein, able to function both as an inhibitory subunit of the Ppz1 protein phosphatase and as a constituent protomer of an unprecedented heterotrimeric PPCDC (phosphopantothenoylcysteine decarboxylase), the third enzyme of the CoA biosynthetic pathway. In the present study we initiated the dissection of the structural elements required for both disparate cellular tasks by using a combination of biochemical and genetic approaches. We show that the conserved Hal3 core [PD (PPCDC domain)] is necessary for both functions, as determined by in vitro and in vivo assays. The Hal3 NtD (N-terminal domain) is not functional by itself, although in vitro experiments indicate that when this domain is combined with the core it has a relevant function in Hal3's heteromeric PPCDC activity. Both the NtD and the acidic CtD (C-terminal domain) also appear to be important for Hal3's Ppz1 regulatory function, although our results indicate that the CtD fulfils the key role in this regard. Finally, we show that the introduction of two key asparagine and cysteine residues, essential for rnonofunctional PPCDC activity but absent in Ha13, is not sufficient to convert it into such a homomeric PPCDC, and that additional modifications of Hal3's PD aimed at increasing its resemblance to known PPCDCs also fails to introduce this activity. This suggests that Hal3 has undergone significant evolutionary drift from ancestral PPCDC proteins. Taken together, our work highlights specific structural determinants that could be exploited for full understanding of Hal3's cellular functions. |
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