Structural Homology to Other Sugar Transporters

The MAL61 gene of Saccharomyces cerewisiae encodes maltose permease, a protein required for the transport of maltose across the plasma membrane. Here we report the nucleotide sequence of the cloned MAL61 gene. A single 1842 bp open reading frame is present within this region encoding the 6 14 residue putative MAL61 protein. Hydropathy analysis suggests that the secondary structure consists of two blocks of six transmembrane domains separated by an approximately 71 residue intracellular region. The N-terminal and C-terminal domains of 100 and 67 residues in length, respectively, also appear to be intracellular. Significant sequence and structural homology is seen between the MAL61 protein and the Saccharomyces high-affinity glucose transporter encoded by the SNF3 gene, the Kluyueromyces lactis lactose permease encoded by the LAC12 gene, the human HepG2 glucose transporter and the Escherichia coli xylose and arabinose transporters encoded by the xylE and araE genes, indicating that all are members of a family of sugar transporters and are related either functionally or evolutionarily. A mechanism for glucose-induced inactivation of maltose transport activity is discussed. M ALTOSE fermentation in the Saccharomyces yeasts is initiated by the transport of the disaccharide across the plasma membrane. This transport is carried out by maltose permease and the process is the rate limiting step in fermentation. An understanding of the mechanisms controlling maltose transport is therefore fundamental to an understanding of the factors regulating maltose fermentation. The Saccharomyces maltose uptake system is an inducible active transport system (HARRIS and THOMPSON 1961 ; OKADA and HALVORSON 1964; DE KROON and KONINGSBERGER 1970; SERRANO 1977). SERRANO (1977) reports that this transport is independent of intracellular ATP levels but is coupled to the electrochemical gradient of protons. That is, maltose transport occurs via a proton symport system. As has been seen in the glucose and galactose transport systems of Saccharomyces, the maltose transport system exists in both a high and a low affinity form (BISSON and FRAENKEL 1983a,b, 1984; RAMOS, SZKUTNICKA and CIRILLO 1989; BUSTURIA and LAGUNAS 1985). The basis of the difference between the two forms of these sugar transporters is not understood. Saccharomyces strains able to ferment maltose carry any one of five MAL loci: MALI, MAL2, MAL3, MAL4, and MAL6 (reviewed by BARNETT 1976). The first of page charges. This article must therefore be hereby marked “advertisement” The publication costs of this article were partly defrayed by the payment in accordance with 18 U.S.C. $1734 solely to indicate this fact. Genetics 123: 477-484 (November, 1989) indication that the gene encoding maltose permease mapped to any of the MAL loci came from the identification of a MALI-linked temperature-sensitive maltose transport mutation (GOLDENTHAL, COHEN and MARMUR 1983). All of the MAL loci have been cloned and structurally and functionally compared (FEDEROFF et al. 1982; NEEDLEMAN and MICHELS 1983; CHARRON, DUBIN and MICHELS 1986; CHARRON and MICHELS 1987; CHARRON et al. 1989). The MAL loci are all highly sequence-homologous, exhibiting only a few restriction site polymorphisms. Each locus is a complex locus containing three genes required for maltose fermentation: GENEs 1, 2, and 3 (NEEDLEMAN et al. 1984). We have established a two digit numbering system in order to distinguish the GENE 1, 2 or 3 functions mapping to the different MAL loci. The first digit indicates the locus position and the second the GENE function (NEEDLEMAN et al. 1984; CHARRON and MICHELS 1987, 1988). Thus, the MAL61 gene is the GENE 1 function mapping to the MAL6 locus. Transcription of GENEs 1 and 2 is induced by maltose and repressed by glucose (NEEDLEMAN et al. 1984). That GENE 2 encodes maltase is inferred from the identification of an allele of the MAL12 gene (that is, GENE 2 of the MALI locus) that encodes a temperature-sensitive maltase (DUBIN et al. 1985). GENE 1 encodes maltose permease. This conclusion is based on several lines of evidence reported by Y. S. CHANG, 478 Q. Cheng and C. A. Michels R. A. DUBIN, E. PERKINS, C. A. MICHELS and R. B. NEEDLEMAN (unpublished r sults). Point mutations in the MAL61 gene as well as a deletion/disruption of the MAL61 gene completely abolish maltose transport activity. Transformation of these mutant strains with high copy plasmids carrying the MAL61 gene leads to up to a tenfold increase in maltose permease activity as compared to the single-copy parental strain. Most significantly, the integration of a fragment carrying the yeast URA3 gene into the coding region f MAL61 near the N-terminal end results in a low level constitutive transcription of MAL61 and in a low level constitutive synthesis of maltose permease. GENE 3 encodes the MAL activator and the product of this gene is a cysteine-zinc finger protein (CHANG et al. 1988; KIM and MICHELS 1988; SOLLITI and MARMUR 1988). This report presents the sequence of the MAL61 gene. Analysis of the deduced amino acid sequence of the proposed MAL61 protein indicates that it is an integral membrane protein. Additionally, MAL61 protein shows significant homology to several other sugar transport proteins from yeast and other species. This homology is seen both on the level of the primary sequence and on the level of secondary structure. MATERIALS AND METHODS Sequencing: Figure 1 shows a restriction endonuclease map of the MAL61 gene. Sequencing was done according to the method of SANGER, NICKLEN and COULSON (1977). The region was divided into three fragments: the PstI-EcoRI fragment containing the MAL61 upstream sequences and the 5’-end of the gene; the 1.7-kb EcoRI-Sal1 fragment containing sequences internal to the MAL61 gene; and the SalI-Hind111 fragment containing the 3’-end of the gene. Each of these was then sequenced by a combination of methods. Nested deletions within the MAL61-insert fragments were constructed with the fragment cloned into the M 13 sequencing vector mpl8 using exonuclease 111 and these were sequenced using the universal primer (MESSING 1983; HENIKOFF 1984). Gaps were filled by using oligonucleotide primers identical to known sequences. Nested deletions were also constructed using Ba131 to degrade the MAL61-insert fragment cloned in the plasmid vector pBR325. For sequencing, these deletions were subcloned into the M 13 sequencing vectors. Sequencing of the second strand was carried out using the 3.6-kb BglII-Hind111 fragment containing the entire MAL61 gene cloned into the M13 vector mp19. This was sequenced with a series of oligonucleotide primers complementary to known MAL61 sequence. Computer analysis: Sequence data were analyzed using the programs of IntelliGenetics, Inc. of Palo Alto, California. Alignment of the MAL61 protein sequence with several other transport protein sequences was carried out using the GENALIGN program. GENALIGN is a copyrighted software product of IntelliGenetics, Inc.; the program was developed by HUGO MARTINEZ of the University of California at San Francisco. The hydropathy plots shown in Figure 4 comparing MAL61 and SNF3 proteins are the gift ofJOHN CELENZA, LINDA MARSHALL-CARLSON and MARIAN CARLSON of the Department of Genetics and Development, Columbia University College of Physicians and Surgeons, New York; the profiles were made using the algorithm developed by KYTE and DOOLITTLE (1 982) and utilized the values of EISENBERG (1 984) with a 2 1 -residue window.