TheSchizosaccharomyces pombe spo3⁺Gene Is Required for Assembly of the Forespore Membrane and Genetically Interacts withpsy1⁺-encoding Syntaxin-like Protein

Abstract

Formation of the forespore membrane, which becomes the plasma membrane of spores, is an intriguing step in the sporulation of the fission yeast Schizosaccharomyces pombe. Here we report two novel proteins that localize to the forespore membrane. spo3 ϩ encodes a potential membrane protein, which was expressed only during sporulation. Green fluorescent protein (GFP) fusion revealed that Spo3 localized to the forespore membrane. The spo3 disruptant was viable and executed meiotic nuclear divisions as efficiently as the wild type but did not form spores. One of the spo3 alleles, spo3-KC51, was dose-dependently suppressed by psy1 ϩ , which encodes a protein similar to mammalian syntaxin-1A, a component of the plasma membrane docking/fusion complex. psy1 ϩ was essential for vegetative growth, and its transcription was enhanced during sporulation. As expected, Psy1 localized to the plasma membrane during vegetative growth. Interestingly, Psy1 on the plasma membrane disappeared immediately after first meiotic division and relocalized to the forespore membrane as the second division initiated. In the spo3 null mutant, the forespore membrane was initiated but failed to develop a normal morphology. Electron microscopy revealed that membrane vesicles were accumulated in the cytoplasm of immature spo3⌬ asci. These results suggest that Spo3 is a key component of the forespore membrane and is essential for its assembly acting in collaboration with the syntaxin-like protein.

Identification of the fnx1+ and fnx2+ genes for vacuolar amino acid transporters in Schizosaccharomyces pombe

“…To tag the Fnx1p and Fnx2p proteins with green fluorescent protein (GFP), each ORF was amplified by PCR and subcloned into pTN197, a derivative of the thiamine-repressible expression vector pREP41 [13], resulting in pTN197/fnx1 and pTN197/fnx2. Cells transformed with these plasmids were examined with an Olympus BX-60 fluorescence microscope using a U-MGFPHQ filter.…”

Live Observation of Forespore Membrane Formation in Fission Yeast

“…To address the molecular mechanism of sporulation, we have identified and analyzed a number of sporulation-specific genes (Bresch et al, 1968;Ikemoto et al, 2000;Asakawa et al, 2001;Nakamura et al, 2001Nakamura et al, , 2002Nakamura et al, , 2005Nakase et al, 2001Nakase et al, , 2004Nakase et al, , 2008Nakamura-Kubo et al, 2003;Yoshida et al, 2005;Ye et al, 2007). Spo2, Spo13, and Spo15 proteins localize to the SPB.…”

“…Anucleated spores are often observed in the spo3 mutant, because FSM formation is impaired (Hirata and Shimoda, 1992;Nakamura et al, 2001). In addition to these genes, the general secretion apparatus is also required for vesicle transport during sporulation.…”

Vsl1p cooperates with Fsv1p for vacuolar protein transport and homotypic fusion in Schizosaccharomyces pombe

“…The vsl1 + ORF was amplified by PCR with SalI and BamHI sites at the ends of DNA fragment. The PCR product was digested with SalI and BamHI, and introduced into the corresponding site of pTN54 derived from pREP41 (Nakamura et al, 2001). Sac.…”

“…pombe cells were observed with an Olympus BX-60 fluorescence microscope (Olympus), and images were captured with a Sensys Cooled CCD camera using the software MetaMorph (Roper Scientific). To observe the localization of Vsl1p, cells were grown to early exponential phase, and fixed with glutaraldehyde and paraformaldehyde as described before (Nakamura et al, 2001). The vacuoles of fission yeast WT and mutant cells were labelled with FM4-64 (Molecular Probes; Vida & Emr, 1995) as described previously (Iwaki et al, 2003).…”