Yolanda Sánchez

Subcellular localization and glycoprotein nature of the invertase from the fission yeast Schizosaccharomyces pombe

The subcellular localization of the enzyme invertase in Schizosaccharomyces pombe cells, both repressed and derepressed for synthesis of the enzyme, was studied. Most of the invertase was found to be located outside the plasma membrane and only a small percentage was found to be associated to membranes. A substantial portion of the external enzyme remained firmly bound to cell-wall material.All of the invertase recovered in soluble form from cellular extracts reacted with concanavalin A and with the lectin from Bandeiraea simplicifolia seeds, indicating the presence in the enzyme of a carbohydrate moiety which probably contains terminal mannosyl (or structurally related) and galactosyl residues.The possibility of the presence of two different forms of invertase in S. pombe was considered. An intracellular, soluble form of invertase, devoid of carbohydrate, similar to the small invertase of the budding yeast Saccharomyces cerevisiae, was not found in S. pombe. However, the Michaelis constant for sucrose of the enzyme present in repressed cells was smaller than that of the invertase synthesized under derepressing conditions, although this difference could also be the result of a different pattern of glycosylation of the invertase synthesized under different growth conditions.

Schizosaccharomyces pombe Rgf3p is a specific Rho1 GEF that regulates cell wall beta-glucan biosynthesis through the GTPase Rho1p.

Rho1p regulates cell integrity by controlling the actin cytoskeleton and cell-wall synthesis. Here, we describe the cloning and characterization of rgf3+, a member of the Rho family of guanine nucleotide exchange factors (Rho GEFs). The rgf3+ gene was cloned by complementation of a mutant (ehs2-1) hypersensitive to drugs that interfere with cell-wall biosynthesis. The rgf3+ gene was found to be essential for cell viability and depletion of Rgf3p afforded phenotypes similar to those obtained following depletion of Rho1p. However, the cell death caused by Rgf3p depletion could be rescued by the presence of 1.2 M sorbitol, whereas depletion of Rho1 was lethal under the same conditions. We show that Rgf3p is a specific Rho1-GEF. The hypersensitivity to drugs affecting the cell wall of the ehs2-1 mutant was suppressed by overexpression of rho1+ but not by any of the other GTPases of the Rho family. Rgf3p interacted with the GDP-bound form of Rho1p and promoted the GDP-GTP exchange. In addition, we show that overexpression of Rgf3p produces multiseptated cells and increases β-1,3-glucan synthase activity and the amount of cell wall β-1,3-glucan. Rgf3p localized to the septum and the mRNA level was regulated in a cell-cycle-dependent manner peaking during septation. Our results suggest that Rgf3p acts as a positive activator of Rho1p, probably activating the Rho functions that coordinate cell-wall biosynthesis to maintain cell integrity during septation.

Synthesis of Saccharomyces cerevisiae invertase by Schizosaccharomyces pombe.

In order to gain information on the ability of the glycosylation system of Schizosaccharomyces pombe to process heterologous glycoproteins, the expression of Saccharomyces cerevisiae invertase in the former yeast was studied. Sc. pombe cells are able to produce enzymatically active invertase from the S. cerevisiae SUC2 gene introduced by transformation and the enzyme is glycosylated and secreted into the cell wall. However, Sc. pombe transformants do not glycosylate the heterologous enzyme as their own invertase since it is not bound by the lectin from Bandeiraea simplicifolia seeds, which indicates the absence of terminal galactose residues. Moreover, the electrophoretic mobility of the heterologous invertase is similar to that of the large enzyme from S. cerevisiae, both in its native form and after being deglycosylated with Endo H. These results suggest that the polypeptide chain of S. cerevisiae invertase is the primary factor for the glycosylation in Sc. pombe cells.

The Putative Exchange Factor Gef3p Interacts with Rho3p GTPase and the Septin Ring during Cytokinesis in Fission Yeast

Background: Guanine nucleotide exchange factors (GEFs) regulate the activity of small GTPases to control cellular functions. Results: Gef3p interacts with the GTPase Rho3p at the division site and localizes to the septin ring. Conclusion: The module Gef3p/Rho3p contributes to downstream events in the secretory pathway. Significance: The interactions between septins and Rho-GEFs provide a new targeting mechanism for GTPases to direct secretion in cytokinesis. The small GTP-binding proteins of the Rho family and its regulatory proteins play a central role in cytokinetic actomyosin ring assembly and cytokinesis. Here we show that the fission yeast guanine nucleotide exchange factor Gef3p interacts with Rho3p at the division site. Gef3p contains a putative DH homology domain and a BAR/IMD-like domain. The protein localized to the division site late in mitosis, where it formed a ring that did not constrict with actomyosin ring (cytokinetic actomyosin ring) invagination; instead, it split into a double ring that resembled the septin ring. Gef3p co-localized with septins and Mid2p and required septins and Mid2p for its localization. Gef3p interacts physically with the GTP-bound form of Rho3p. Although Gef3p is not essential for cell separation, the simultaneous disruption of gef3+ and Rho3p-interacting proteins, such as Sec8p, an exocyst component, Apm1p, a subunit of the clathrin adaptor complex or For3p, an actin-polymerizing protein, yielded cells with strong defects in septation and polarity respectively. Our results suggest that interactions between septins and Rho-GEFs provide a new targeting mechanism for GTPases in cytokinesis, in this case probably contributing to Rho3p function in vesicle tethering and vesicle trafficking in the later steps of cell separation.

The fission yeast cell wall stress sensor-like proteins Mtl2 and Wsc1 act by turning on the GTPase Rho1p but act independently of the cell wall integrity pathway

Sensing stressful conditions that affect the cell wall reorganization is important for yeast survival. Here, we studied two proteins SpWsc1p and SpMtl2p with structural features indicative of plasma membrane‐associated cell wall sensors. We found that Mtl2p and Wsc1p act by turning on the Rho1p GTPase. Each gene could be deleted individually without affecting viability, but the deletion of both was lethal and this phenotype was rescued by overexpression of the genes encoding either Rho1p or its GDP/GTP exchange factors (GEFs). In addition, wsc1Δ and mtl2Δ cells showed a low level of Rho1p‐GTP under cell wall stress. Mtl2p‐GFP (green fluorescent protein) localized to the cell periphery and was necessary for survival under different types of cell wall stress. Wsc1p‐GFP was concentrated in patches at the cell tips, it interacted with the Rho‐GEF Rgf2p, and its overexpression activated cell wall biosynthesis. Our results are consistent with the notion that cell wall assembly is regulated by two different networks involving Rho1p. One includes signaling from Mtl2p through Rho1p to Pck1p, while the second one implicates signaling from Wsc1p and Rgf2p through Rho1p to activate glucan synthase (GS). Finally, signaling through the mitogen‐activated protein kinase (MAPK) Pmk1p remained active in mtl2Δ and wsc1Δ disruptants exposed to cell wall stress, suggesting that the cell wall stress‐sensing spectrum of Schizosaccharomyces pombe sensor‐like proteins differs from that of Saccharomyces cerevisiae.

The checkpoint–dependent nuclear accumulation of Rho1p exchange factor Rgf1p is important for tolerance to chronic replication stress

Fission yeast Rho1p exchange factor Rgf1p is relocalized to the cell nucleus during the stalled replication caused by hydroxyurea. The Cds1p checkpoint kinase, helped by the chaperone Rad24p, controls the nuclear accumulation of Rgf1p by inhibiting its nuclear export. Failure to do so produces inefficient recovery from replication arrest.

Rgf1p (Rho1p GEF) is required for double-strand break repair in fission yeast

Abstract Rho GTPases are conserved molecules that control cytoskeletal dynamics. These functions are expedited by Rho GEFs that stimulate the release of GDP to enable GTP binding, thereby allowing Rho proteins to initiate intracellular signaling. How Rho GEFs and Rho GTPases protect cells from DNA damage is unknown. Here, we explore the extreme sensitivity of a deletion mutation in the Rho1p exchange factor Rgf1p to the DNA break/inducing antibiotic phleomycin (Phl). The Rgf1p mutant cells are defective in reentry into the cell cycle following the induction of severe DNA damage. This phenotype correlates with the inability of rgf1Δ cells to efficiently repair fragmented chromosomes after Phl treatment. Consistent with this observation Rad11p (ssDNA binding protein, RPA), Rad52p, Rad54p and Rad51p, which facilitate strand invasion in the process of homology-directed repair (HDR), are permanently stacked in Phl-induced foci in rgf1Δ cells. These phenotypes are phenocopied by genetic inhibition of Rho1p. Our data provide evidence that Rgf1p/Rho1p activity positively controls a repair function that confers resistance against the anti-cancer drug Phl.