Characterization of a new multigene family encoding isomaltases in the yeast Saccharomyces cerevisiae, the IMA family

Abstract

It has been known for a long time that the yeast Saccharomyces cerevisiae can assimilate alpha-methylglucopyranoside and isomaltose. We here report the identification of 5 genes (YGR287c, YIL172c, YJL216c, YJL221c and YOL157c), which, similar to the SUCx, MALx, or HXTx multigene families, are located in the subtelomeric regions of different chromosomes. They share high nucleotide sequence identities between themselves (66-100%) and with the MALx2 genes (63-74%). Comparison of their amino acid sequences underlined a substitution of threonine by valine in region II, one of the four highly conserved regions of the alpha-glucosidase family. This change was previously shown to be sufficient to discriminate alpha-1,4- to alpha-1,6-glucosidase activity in YGR287c (Yamamoto, K., Nakayama, A., Yamamoto, Y., and Tabata, S. (2004) Eur. J. Biochem. 271, 3414-3420). We showed that each of these five genes encodes a protein with alpha-glucosidase activity on isomaltose, and we therefore renamed these genes IMA1 to IMA5 for IsoMAltase. Our results also illustrated that sequence polymorphisms among this family led to interesting variability of gene expression patterns and of catalytic efficiencies on different substrates, which altogether should account for the absence of functional redundancy for growth on isomaltose. Indeed, deletion studies revealed that IMA1/YGR287c encodes the major isomaltase and that growth on isomaltose required the presence of AGT1, which encodes an alpha-glucoside transporter. Expressions of IMA1 and IMA5/YJL216c were strongly induced by maltose, isomaltose, and alpha-methylglucopyranoside, in accordance with their regulation by the Malx3p-transcription system. The physiological relevance of this IMAx multigene family in S. cerevisiae is discussed.

FIGURE 1.

Four conserved regions of the GH13 family. ClustalW amino acid sequence alignment of MAL12, MAL32, and five unlinked genes encoding putative α-glucosidase, which have been renamed IMA1 through IMA5. The three most important catalytic residues are written in boldface and marked with a star. The arrows indicate the position of the amino acid residue that discriminates α-1,6- (Val) from α-1,4- (Thr) linkages of glucosidic substrates.

FIGURE 2.

Biochemical activities on α-1,6- and α-1,4-glucosidic substrates. The α-glucosidase activity on different α-glucosidic substrates was assayed on crude extract from JF1811 yeast strain overexpressing MAL12 or ORFs of this gene family. ★, because IMA3 and IMA4 are 100% identical, they could not be differentiated. The cells were grown in YN synthetic medium containing 2% glucose to A_{600 nm} ∼ 1.0, and activities were measured using 20 A_{600 nm} units of cells. Control: JF1811 strain transformed with the empty YEplac181-PGK/CYC1 plasmid. The α-p-nitrophenyl α-d-glucopyranoside (pNPG) was used to measure total α-glucosidase activity. Maltose and maltotriose were used to measure maltase activity. αMG and isomaltose were used to measure isomaltase activity. Activities and standard deviation were calculated from three independent cultures.

FIGURE 3.

Qualitative growth assay of deletion mutants and overexpression strains on isomaltose and αMG. The yeast strains were grown on YN liquid medium containing 1% sugar (isomaltose or αMG). A, growth of the wild-type strain CEN.PK113–7D, its five isogenic single deletion mutants from this new gene family, and its agt1Δ derivative (MATa MAL2–8^c SUC2 agt1::lacZ loxP-kanMX4-loxP (66)). The cultivation tubes were incubated at 30 °C for 24 h prior to being photographed. B, the ygr287cΔ leu2Δ mutant strain was transformed by plasmids overexpressing the different ORFs of this gene family. Control: empty YEplac181-PGK/CYC1 plasmid. The tubes were photographed when cells reached the stationary phase after 72 h of incubation at 30 °C. Normalized values of A_{600 nm} (±2 S.E., see “Experimental Procedures”), after 24 h of cultivation on isomaltose and αMG, were respectively: 1.0 ± 0.14 and 1.0 ± 0.27 (IMA1); 0.95 ± 0.11 and 0.88 ± 0.20 (IMA2); 0.69 ± 0.13 and 0.25 ± 0.06 (IMA3*); 0.76 ± 0.08 and 0.008 ± 0.002 (IMA5); and 0.09 ± 0.03 and 0.02 ± 0.01 (empty plasmid). ★, IMA3 or IMA4.

FIGURE 4.

Qualitative growth assay of yeast strains harboring different Mal phenotypes on maltose, isomaltose, and αMG. The wild-type CEN.PK113–7D strain (Mal⁺) and its MAL2–8^c deletion mutant, the wild-type BY4741 strain (Mal⁻), and the BY4741 strain transformed with a CEN plasmid bearing the constitutively expressed MAL2–8^c allele of the MAL23 gene, were grown on YN liquid medium containing 1% sugar (maltose, isomaltose, or αMG). The cultivation tubes were incubated at 30 °C for 24 h prior to being photographed.

FIGURE 5.

Putative binding sites of transcriptional regulators. The 700-bp sequences upstream of the start codon of MAL12, MAL32, and AGT1 and the five IMAx genes were analyzed for consensus binding motifs using the YEASTRACT database (available on-line) and using the fuzznuc program for TATA box patterns searching (Mobyle database, available on-line). TATA Box: TATAA/TAA/TA/G (■) (60); Msn2/4p binding site (STRE consensus): CCCCT (□) (43); Mal63p binding site: MGCN{9}MGS (●) (67); Mig1p binding site: ATTTTTGTGGGG (among 12 published consensus) (◆) (68).