Comparative Analysis of Type 1 and Type Z Protein Phosphatases Reveals D615 as a Key Residue for Ppz1 Regulation

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.

Functional mapping of the N‐terminal region of the yeast moonlighting protein Sis2/Hal3 reveals crucial residues for Ppz1 regulation

“…We show here that, indeed, this region is vital for regulation of Ppz1. This is in keeping with the very recent evidence obtained by chemical crosslinking showing that the vast majority of interactions detected between the catalytic C-terminal domain of Ppz1 and Hal3 involve residues located within this Nterminal extension [33].…”

“…1A ). These specific regions were selected based on two criteria: (a) sequence conservation among different yeast species, and (b) the results of our recently reported chemical cross‐linking experiments [ 33 ], which suggested several interactions between the catalytic C‐terminal domain of Ppz1 and diverse regions within the N‐terminal Hal3 extension. These versions were cloned in plasmid pWS93, to allow multicopy expression from the ADH1 promoter, and were subsequently introduced into diverse yeast strains to test for the function of these Hal3 versions as Ppz phosphatase regulators.…”

When Phosphatases Go Mad: The Molecular Basis for Toxicity of Yeast Ppz1

“…As mentioned above, the role of Hal3 in salt tolerance and cell cycle regulation [71,72] was explained by its identification as a protein able to bind to Ppz1 at its C-terminal catalytic domain and inhibit its phosphatase activity [21,46]. However, in spite of considerable effort, the molecular basis for this inhibitory mechanism is not fully understood [73][74][75][76][77][78].…”

Fungal Hal3 (and Its Close Relative Cab3) as Moonlighting Proteins

“…Hal3 can be recovered bound to Ppz1 when both proteins are simultaneously expressed in E. coli [ 39 ]. This fact was exploited to carry out chemical cross-linking experiments, involving full Hal3 and the C-terminal catalytic domain of Ppz1, followed by the identification of the covalently bound peptides by mass spectrometry [ 41 ]. It was found that most Hal3 links were restricted to the N-terminal half and the first third of the conserved PD domain.…”