Role of the α-Glucanase Agn1p in Fission-Yeast Cell Separation

Abstract

Cell division in the fission yeast Schizosaccharomyces pombe yields two equal-sized daughter cells. Medial fission is achieved by deposition of a primary septum flanked by two secondary septa within the dividing cell. During the final step of cell division, cell separation, the primary septum is hydrolyzed by an endo-(1,3)-␤-glucanase, Eng1p. We reasoned that the cell wall material surrounding the septum, referred to here as the septum edging, also must be hydrolyzed before full separation of the daughter cells can occur. Because the septum edging contains (1,3)-␣-glucan, we investigated the cellular functions of the putative (1,3)-␣-glucanases Agn1p and Agn2p. Whereas agn2 deletion results in a defect in endolysis of the ascus wall, deletion of agn1 leads to clumped cells that remained attached to each other by septum-edging material. Purified Agn1p hydrolyzes (1,3)-␣-glucan predominantly into pentasaccharides, indicating an endo-catalytic mode of hydrolysis. Furthermore, we show that the transcription factors Sep1p and Ace2p regulate both eng1 and agn1 expression in a cell cycle-dependent manner. We propose that Agn1p acts in concert with Eng1p to achieve efficient cell separation, thereby exposing the secondary septa as the new ends of the daughter cells.

Role of the α‐glucanase Agn2p in ascus‐wall endolysis following sporulation in fission yeast

“…pombe strains agn2 strains were described previously (Dekker et al, 2004). Diploid strains ND080 (h − /h + ade6-M210 /ade6-M216 ) and ND088 (h − /h + agn2 ::kanMX4 /agn2 ::kanMX4 ade6-M210 /ade6-M216 ) were constructed by conjugating strains FYC11 (h − ade6-M210 ) with FYC15 (h + ade6-M216 ) and strains ND044 (h − agn2 ::kanMX4 ade6-M210 ) with ND047 (h + agn2 ::kanMX4 ade6-M216 ), respectively, and by selecting for adenine prototrophy using the complementing chromosomal alleles ade6-M210 and ade6-M216.…”

“…Diploid strains ND080 (h − /h + ade6-M210 /ade6-M216 ) and ND088 (h − /h + agn2 ::kanMX4 /agn2 ::kanMX4 ade6-M210 /ade6-M216 ) were constructed by conjugating strains FYC11 (h − ade6-M210 ) with FYC15 (h + ade6-M216 ) and strains ND044 (h − agn2 ::kanMX4 ade6-M210 ) with ND047 (h + agn2 ::kanMX4 ade6-M216 ), respectively, and by selecting for adenine prototrophy using the complementing chromosomal alleles ade6-M210 and ade6-M216. For overexpression of agn2 from its chromosomal locus, a kanMX6-Pnmt1 cassette was integrated in front of the agn2 ORF of wild-type strain 972 (h − ) by using a PCR-mediated strategy (Bähler et al, 1998) with minor modifications (Dekker et al, 2004), using primers ND161 (5 -TTGTATAGCGGCAGTCATCGGCTAGACCTT-TGAAAGCGGACAGCTTGCATTTCCACTAAC-CAACTTAAATATCACTTAGAATTCGAGCTC-GTTTAAACTG) and ND162 (5 -AGCATAATTG-TACGTAAGACCCATCATAAAATGGGCTACA-ACGGCTTTATTCGGTAAAGCTGTTGTTAAC-GACGCCATGTTTAACAAAGCGACTATAAG). Using several mating steps together with diploid selection, diploid strain ND227 (h − /h + agn2::Pnmt1-kanMX6 /agn2 ::kanMX4 ade6-M210 /ade6-M216 ) was generated.…”

“…Fractions with the highest (1,3)-α-glucanase activity were used for further study. Purity was monitored by SDS-PAGE analysis as described previously (Dekker et al, 2004).…”

“…Interestingly, the gene encoding the paralogue of the characterized endo-(1,3)-α-glucanase Agn1p, Agn2p, is also highly upregulated during the middle phase (Mata et al, 2002). Recently, we showed that, although also expressed during vegetative growth, agn2 + appears to function during sporulation (Dekker et al, 2004). Agn2p-GFP localized to the epiplasm, the residual cytoplasm of the diploid cell, which surrounds the ascospores inside the ascus wall (Yoo et al, 1973).…”

“…We show that expression of agn2 is required for efficient dispersal of ascospores. Because Agn2p shares almost 40% amino acid sequence identity with Agn1p (Dekker et al, 2004), we investigate the enzyme characteristics of this putative α-glucanase. Finally, we show that remnants of the ascus wall of agn2 /agn2 asci are hydrolysed upon incubation with purified Agn2p-his, releasing the individual ascospores into the culture medium.…”