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. 2005 Dec;17(12):3532–3543. doi: 10.1105/tpc.105.036285

Figure 1.

Figure 1.

Identification of gas1 as a Gene Encoding an α-Subunit of Glucosidase II.

(A) Schematic representation of the pSMUT insertion in gas1 in mutant strain CL13pat4339. The open reading frame of Gas1 is indicated by an open arrow. The neighboring predicted open reading frames are indicated by gray arrows, which are interrupted at potential intron positions (MUMDB, http://mips.gsf.de/genre/proj/ustilago/; shown are open reading frames um04407, um10516, um04405, and um04404 from contig 1.157). Relevant restriction enzyme sites are indicated by letters: B, BamHI, M, MluI; N, NcoI, P, PstI. The pSMUT insertion took place in a genomic BamHI site located in the 3′ region of the 3183-bp open reading frame at bp 2642. The restriction sites used for cloning are indicated by asterisks above the respective sites. The relative positions of the two partial cDNA clones pcgas1 and pcgas2 are indicated by black lines below the figure. The pSMUT insertion is not drawn to scale.

(B) Gas1 shares significant sequence similarity with the putative α-glucosidases from N. crassa (Nc, CAD36981), S. pombe (Sc, CAB65603), D. discoideum (Dd, ModA; AAB18921), and M. musculus (Mm, AAC53182). Only the region around the catalytic site is shown. Identical amino acids are highlighted by gray shading. Gas1 is predicted to carry a cleavable N-terminal signal sequence (amino acids 1 to 24; data not shown) and possesses the characteristic catalytic site of glycosyl hydrolases ([(G/F)-(L/I/V/M)-W-X-D-M-N-E], indicated by asterisks above the sequence).

(C) Gas1 complements the temperature-sensitive phenotype of a S. cerevisiae rot1-1 rot2-1 double mutant. S. cerevisiae MH272-1da rot1-1 rot2-1/pLeu cells transformed with either empty vector (pYES2) or the pYES2 derivatives containing either the yeast ROT2 gene encoding α-glucosidase II (pRot2) or the U. maydis gas1 gene (pGas1) under the control of a galactose-inducible promoter were streaked on plates containing glucose (YPglc) or galactose (YPgal) and incubated at 28 and 37°C. rot1-1 rot2-1 mutants are viable at 37°C if they express Rot2p or U. maydis Gas1.