Molecular Characterization of Ypi1, a Novel Saccharomyces cerevisiae Type 1 Protein Phosphatase Inhibitor

Abstract

The Saccharomyces cerevisiae open reading frame YFR003c encodes a small (155-amino acid) hydrophilic protein that we identified as a novel, heat-stable inhibitor of type 1 protein phosphatase (Ypi1). Ypi1 interacts physically in vitro with both Glc7 and Ppz1 phosphatase catalytic subunits, as shown by pull-down assays. Ypi1 inhibits Glc7 but appears to be less effective toward Ppz1 phosphatase activity under the conditions tested. Ypi1 contains a 48 RHNVRW 53 sequence, which resembles the characteristic consensus PP1 phosphatase binding motif. A W53A mutation within this motif abolishes both binding to and inhibition of Glc7 and Ppz1 phosphatases. Deletion of YPI1 is lethal, suggesting a relevant role of the inhibitor in yeast physiology. Cells overexpressing Ypi1 display a number of phenotypes consistent with an inhibitory role of this protein on Glc7, such as decreased glycogen content and an increased growth defect in a slt2/mpk1 mitogen-activated protein kinase-deficient background. Taking together, these results define Ypi1 as the first inhibitory subunit of Glc7 identified in budding yeast.

Protein factors in pre-mRNA 3′-end processing

“…Depletion, inhibition or removal of Glc7p results in mRNAs with shortened poly(A) tails, indicating that Glc7p plays a role in polyadenylation but not cleavage [204,230]. The level of phosphorylated Pta1p increases upon depletion of Glc7p in vivo, identifying Pta1p as a specific target for Glc7p phosphotase activity.…”

Protein phosphatase CaPpz1 is involved in cation homeostasis, cell wall integrity and virulence of Candida albicans

“…The phosphatase activity of the fractions was assayed in triplicate samples containing 1-2 mg recombinant phosphatase with p-nitrophenylphosphate (Sigma-Aldrich) substrate in the presence or absence of Hal3, as reported previously (Muñoz et al, 2004). Recombinant S. cerevisiae Hal3 was expressed in E. coli and purified according to García-Gimeno et al (2003).…”

Functional Characterization of the Yeast Ppz1 Phosphatase Inhibitory Subunit Hal3

“…GST-Ppz1⌬ 1-344 was expressed in bacteria and bound to glutathione-agarose beads essentially as described previously (21). Yeast extracts of strain IM021 (ppz1 hal3) transformed with the YEplac195 multicopy plasmids carrying the different versions of Hal3 under study were basically prepared as described by de Nadal et al (9).…”

“…To this end, the entire open reading frame of the different HAL3 versions was amplified by PCR using oligonucleotides 5Ј-HAL3EcoRI and 3Ј-HAL3 XhoI to generate a 1.7-kbp fragment that was then cloned into these same sites of plasmid pGEX6P-1 (Amersham Biosciences). The different versions of GSTHal3 generated were expressed using 1 mM isopropyl-1-thio-␤-D-galactopyranoside for induction at 37°C for 3 h. Conditions of expression and purification of bacterial recombinant GST-Ppz1 ⌬1-344 were essentially as described previously (21). Once bound to the glutathione-agarose affinity column, the recombinant phosphatase was treated for 4 h at 4°C with PreScission protease (Amersham Biosciences) following the manufacturer's indications (80 units/ml resin) to cleave the GST moiety.…”

“…In contrast, besides the early observation that the characteristic highly acidic tail of Hal3 is required for function in vivo (7,8), very little is known regarding the structural elements defining the cellular role of Hal3 or its function as phosphatase inhibitor. Interestingly, Hal3 does not structurally resemble previously characterized PP1c phosphatase inhibitors and it does not bind or inhibit in vitro yeast PP1c (encoded by GLC7 in S. cerevisiae) (9,21). Therefore, previous experience and knowledge on the regulation of Glc7 by its diverse regulatory subunits were of little help to face the question of how Hal3 might bind to and inhibit Ppz1.…”