We have recently characterized Ypi1 as an inhibitory subunit of yeast Glc7 PP1 protein phosphatase. In this work we demonstrate that Ypi1 forms a complex with Glc7 and Sds22, another Glc7 regulatory subunit that targets the phosphatase to substrates involved in cell cycle control. Interestingly, the combination of equimolar amounts of Ypi1 and Sds22 leads to an almost full inhibition of Glc7 activity. Because YPI1 is an essential gene, we have constructed conditional mutants that demonstrate that depletion of Ypi1 leads to alteration of nuclear localization of Glc7 and cell growth arrest in mid-mitosis with aberrant mitotic spindle. These phenotypes mimic those produced upon inactivation of Sds22. The fact that progressive depletion of either Ypi1 or Sds22 resulted in similar physiological phenotypes and that both proteins inhibit the phosphatase activity of Glc7 strongly suggest a common role of these two proteins in regulating Glc7 nuclear localization and function.

It was shown recently that individual cells of an isogenic Saccharomyces cerevisiae population show variability in acetic acid tolerance, and this variability affects the quantitative manifestation of the trait at the population level. In the current study, we investigated whether cell-to-cell variability in acetic acid tolerance could be explained by the observed differences in the cytosolic pHs of individual cells immediately before exposure to the acid. Results obtained with cells of the strain CEN.PK113-7D in synthetic medium containing 96 mM acetic acid (pH 4.5) showed a direct correlation between the initial cytosolic pH and the cytosolic pH drop after exposure to the acid. Moreover, only cells with a low initial cytosolic pH, which experienced a less severe drop in cytosolic pH, were able to proliferate. A similar correlation between initial cytosolic pH and cytosolic pH drop was also observed in the more acid-tolerant strain MUCL 11987-9. Interestingly, a fraction of cells in the MUCL 11987-9 population showed initial cytosolic pH values below the minimal cytosolic pH detected in cells of the strain CEN.PK113-7D; consequently, these cells experienced less severe drops in cytosolic pH. Although this might explain in part the difference between the two strains with regard to the number of cells that resumed proliferation, it was observed that all cells from strain MUCL 11987-9 were able to proliferate, independently of their initial cytosolic pH. Therefore, other factors must also be involved in the greater ability of MUCL 11987-9 cells to endure strong drops in cytosolic pH.

Saccharomyces cerevisiae Hal3 is a conserved protein that binds the carboxyl-terminal catalytic domain of the PP1c (protein phosphatase 1)-related phosphatase Ppz1 and potently inhibits its activity, thus modulating all of the characterized functions so far of the phosphatase. It is unknown how Hal3 binds to Ppz1 and inhibits its activity. Although it contains a putative protein phosphatase 1c binding-like sequence (²⁶³KLHVLF²⁶⁸), mutagenesis analysis suggests that this motif is not required for Ppz1 binding and inhibition. The mutation of the conserved His³⁷⁸ (possibly involved in dehydrogenase catalytic activity) did not impair Hal3 functions or Ppz1 binding. Random mutagenesis of the 228 residue-conserved central region of Hal3 followed by a loss-of-function screen allowed the identification of nine residues important for Ppz1-related Hal3 functions. Seven of these residues cluster in a relatively small region spanning from amino acid 446 to 480. Several mutations affected Ppz1 binding and inhibition in vitro, whereas changes in Glu⁴⁶⁰ and Val⁴⁶² did not alter binding but resulted in Hal3 versions unable to inhibit the phosphatase. Therefore, there are independent Hal3 structural elements required for Ppz1 binding and inhibition. S. cerevisiae encodes a protein (Vhs3) structurally related to Hal3. Recent evidence suggests that both mutations are synthetically lethal. Surprisingly, versions of Hal3 carrying mutations that strongly affected Ppz1 binding or inhibitory capacity were able to complement lethality. In contrast, the mutation of His³⁷⁸ did not. This finding suggests that Hal3 may have both Ppz1-dependent and independent functions involving different structural elements.

Glc7 is the only catalytic subunit of the protein phosphatase type 1 in the yeast S. cerevisiae and, together with its regulatory subunits, is involved in many essential processes. Analysis of the non-essential mutants in the regulatory subunits of Glc7 revealed that the lack of Reg1, and no other subunit, causes hypersensitivity to unfolded protein response (UPR)-inducers, which was concomitant with an augmented UPR element-dependent transcriptional response. The Glc7–Reg1 complex takes part in the regulation of the yeast AMP-activated serine/threonine protein kinase Snf1 in response to glucose. We demonstrate in the present study that the observed phenotypes of reg1 mutant cells are attributable to the inappropriate activation of Snf1. Indeed, growth in the presence of limited concentrations of glucose, where Snf1 is active, or expression of active forms of Snf1 in a wild-type strain increased the sensitivity to the UPR-inducer tunicamycin. Furthermore, reg1 mutant cells showed a sustained HAC1 mRNA splicing and KAR2 mRNA levels during the recovery phase of the UPR, and dysregulation of the Ire1-oligomeric equilibrium. Finally, overexpression of protein phosphatases Ptc2 and Ptc3 alleviated the growth defect of reg1 cells under endoplasmic reticulum (ER) stress conditions. Altogether, our results reveal that Snf1 plays an important role in the attenuation of the UPR, as well as identifying the protein kinase and its effectors as possible pharmacological targets for human diseases that are associated with insufficient UPR activation.

Rheumatic diseases such as osteoarthritis (OA) are a major social and economic burden because of the population aging and the lack of curative solutions. An effective cell therapy may be the best treatment option for OA and other cartilage diseases. However, the main cellular strategy used to repair articular cartilage, the transplantation of autologous chondrocytes, is limited to a small number of patients with traumatic lesions. The use of joint replacement after years of disease progression proves the great medical need in current practice. Mesenchymal stromal/stem cells (MSCs) provide an alternative cell source for cartilage regeneration due to numerous advantages, comprising relative ease to isolate and culture, chondrogenic capacity, and anti-inflammatory effects. Initial clinical trials with MSCs have led to encouraging results, but many variables have to be considered to attain true amelioration of disease or repair (type and status of cartilage disease, source and conditions of cells, administration regime, combinatorial approaches). Particularly, allogeneic MSCs are an advantageous cellular product. The animal models chosen for preclinical evaluation are also relevant for successful translation into clinical practice. Considering the limitations in the field, rigorous comparative and validating studies in well-established animal models (including large animals) are still needed to set up the bases for additional clinical trials. The present review of studies performed in small and large animal models should help clarify the applicability of MSC-based therapies for articular cartilage repair.

Ypi1 was discovered as an essential protein able to act as a regulatory subunit of the Saccharomyces cerevisiae type 1 protein phosphatase Glc7 and play a key role in mitosis. We show here that partial depletion of Ypi1 causes lithium sensitivity and that high levels of this protein confer a lithium-tolerant phenotype to yeast cells. Remarkably, this phenotype was independent of the role of Ypi1 as a Glc7 regulatory subunit. Lithium tolerance in cells overexpressing Ypi1 was caused by a combination of increased efflux of lithium, mediated by augmented expression of the alkaline cation ATPase ENA1, and decreased lithium influx through the Trk1,2 high-affinity potassium transporters. Deletion of CNB1, encoding the regulatory subunit of the calcineurin phosphatase, blocked Ypi1-induced expression of ENA1, normalized Li⁺ fluxes, and abolished the Li⁺ hypertolerant phenotype of Ypi1-overexpressing cells. These results point to a complex role of Ypi1 on the regulation of cation homeostasis, largely mediated by the calcineurin phosphatase.