Abstract
Type 1 Ser/Thr protein phosphatases are represented in all fungi by two enzymes, the ubiquitous PP1, with a conserved catalytic polypeptide (PP1c) and numerous regulatory subunits, and PPZ, with a C-terminal catalytic domain related to PP1c and a variable N-terminal extension. Current evidence indicates that, although PP1 and PPZ enzymes might share some cellular targets and regulatory subunits, their functions are quite separated, and they have individual regulation. We explored the structures of PP1c and PPZ across 57 fungal species to identify those features that (1) are distinctive among these enzymes and (2) have been preserved through evolution. PP1c enzymes are more conserved than PPZs. Still, we identified 26 residues in the PP1 and PPZ catalytic moieties that are specific for each kind of phosphatase. In some cases, these differences likely affect the distribution of charges in the surface of the protein. In many fungi, Hal3 is a specific inhibitor of the PPZ phosphatases, although the basis for the interaction of these proteins is still obscure. By in vivo co-purification of the catalytic domain of ScPpz1 and ScHal3, followed by chemical cross-linking and MS analysis, we identified a likely Hal3-interacting region in ScPpz1 characterized by two major and conserved differences, D566 and D615 in ScPpz1, which correspond to K210 and K259 in ScPP1c (Glc7). Functional analysis showed that changing D615 to K renders Ppz1 refractory to Hal3 inhibition. Since ScHal3 does not regulate Glc7 but it inhibits all fungal PPZ tested so far, this conserved D residue could be pivotal for the differential regulation of both enzymes in fungi.
Highlights
Analysis of the Saccharomyces cerevisiae genome allows the identification of at least 19 proteins with predicted or experimentally tested Ser/Thr protein phosphatase activity
By in vivo co-purification of the catalytic domain of ScPpz1 and ScHal3, followed by chemical cross-linking and MS analysis, we identified a likely Hal3-interacting region in ScPpz1 characterized by two major and conserved differences, D566 and D615 in ScPpz1, which correspond to K210 and K259 in ScPP1c (Glc7)
In contrast to E615, E575 and E630 are conserved in Glc7 (D219 and E274, respectively), and in the vast majority of fungal species analyzed in this work. All these results indicate that the conserved D615 in Ppz1 is an important structural determinant that contributes to the specificity of the regulation of Ppz and PP1c enzymes in fungi
Summary
Analysis of the Saccharomyces cerevisiae genome allows the identification of at least 19 proteins with predicted or experimentally tested Ser/Thr protein phosphatase activity. It is worth noting that whereas in the35c6a/s3e5o9f Ppz1Cter the 16 cross-linked reactive ly3s9ines are widely distributed along the polyp35e8ptide; in the case of Hal, the links ar2e9,m41o,s3tl1y5 restricted to the N-terminal half and3t7h8e first third of the conserved PD do1m13a, 1in97(,t3h1e5,p3a1r6t that shares similarity with known P33P8916CDC enzymes) This is interesting becau2s1e02p,346re8v3 ious work reported that the N-terminal e4x3t3ension of Hal is, by itself, unable to intera2c1t 0in vitro with Ppz (neither with the full-le4n5g3th protein nor with its catalytic domain)2, 8b0ut it is relevant for the inhibitory function46[822]. All these results indicate that the conserved D615 in Ppz is an important structural determinant that contributes to the specificity of the regulation of Ppz and PP1c enzymes in fungi
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