Vacuolar protein sorting in fission yeast: cloning, biosynthesis, transport, and processing of carboxypeptidase Y from Schizosaccharomyces pombe

Abstract

PCR was used to isolate a carboxypeptidase Y (CPY) homolog gene from the fission yeast Schizosaccharomyces pombe. The cloned S. pombe cpy1؉ gene has a single open reading frame, which encodes 950 amino acids with one potential N-glycosylation site. It appears to be synthesized as an inactive pre-pro protein that likely undergoes processing following translocation into appropriate intracellular organelles. The C-terminal mature region is highly conserved in other serine carboxypeptidases. In contrast, the N-terminal pro region containing the vacuolar sorting signal in CPY from Saccharomyces cerevisiae shows fewer identical residues. The pro region contains two unusual repeating sequences; repeating sequence I consists of seven contiguous repeating segments of 13 amino acids each, and repeating sequence II consists of seven contiguous repeating segments of 9 amino acids each. Pulse-chase radiolabeling analysis revealed that Cpy1p was initially synthesized in a 110-kDa pro-precursor form and via the 51-kDa single-polypeptide-chain intermediate form which has had its pro segment removed is finally converted to a heterodimer, the mature form, which is detected as a 32-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Like S. cerevisiae CPY, S. pombe Cpy1p does not require the N-linked oligosaccharide moiety for vacuolar delivery. To investigate the vacuolar sorting signal of S. pombe Cpy1p, we have constructed cpy1

Multiple functions of ergosterol in the fission yeast Schizosaccharomyces pombe

“…pombe were carried out as described by Tabuchi et al (1997). Antibody incubations were carried out using rabbit polyclonal antibody against Schiz.…”

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

“…pombe strains ARC039 (h + leu1 ura4) and KJ100-7B (h 90 leu1 ura4) were provided by Yuko Giga-Hama (Asahi Glass) and Koichi Tanaka (University of Tokyo, Japan). The disruptants cpy1D (h + leu1 ura4-D18 his2 ade6-M216 cpy1 : : ura4), vps34/pik3D (h + leu1 ura4-D18 his2 ade6-M216 vps34/pik3 : : ura4), ste12D (h 90 leu1 ura4-D18 ade6-M210 ste12 : : ura4), fsv1D (h + leu1 ura4-D18 his2 ade6-M216 fsv1 : : ura4) and pep12D (h + leu1 ura4-D18 his2 ade6-M216 pep12 : : ura4) were constructed as described previously (Tabuchi et al, 1997a;Takegawa et al, 1995Takegawa et al, , 2003aOnishi et al, 2003;Hosomi et al, 2011 DNA cloning, gene disruption and plasmid construction. To disrupt the endogenous Sch.…”

“…Pulse-chase and immunoblot analyses of the vacuolar carboxypeptidase Y from Sch. pombe (SpCPY) were performed as described before (Tabuchi et al, 1997a). Antibody reactions were performed using a rabbit polyclonal antibody against SpCPY.…”

“…We previously reported the isolation and characterization of a vacuolar marker protein, carboxypeptidase Y from Sch. pombe (SpCPY) (Tabuchi et al, 1997a). In WT cells, SpCPY is efficiently sorted to the vacuole, and therefore is not secreted through the plasma membrane.…”

Atg20- and Atg24-family proteins promote organelle autophagy in fission yeast

“…To examine whether organelle autophagy occurs in wild-type cells, we applied nitrogen starvation treatment, which is a trigger of general autophagy in S. pombe (Kohda et al, 2007;Mukaiyama et al, 2009;Sun et al, 2013), and monitored the subcellular localization of two organelle markers, an ER integral membrane protein Ost4 and a mitochondrial matrix protein Sdh2. We used Cpy1 as a vacuole lumen marker (Tabuchi et al, 1997). Under nutrient-rich conditions (+N), neither Ost4 nor Sdh2 showed obvious overlap with Cpy1 ( Fig.…”