No abstract

A novel, systematic approach was used to identify amino acid residues responsible for substrate recognition in the transmembrane 10 region of the Gal2 galactose transporter of Saccharomyces cerevisiae. Site-directed mutagenesis has been extensively used in attempts to determine functional sites in transporters (1, 2). This approach is limited, however, by the fact that it is usually not possible to mutate every amino acid and replacements that are made often yield results that are negative in nature (3, 4). As an alternative method, the use of chimeras to identify functional domains of transporters has proved highly fruitful (5-9).We have used chimeras to analyze two homologous sugar transporters in the yeast Saccharomyces cerevisiae (3, 4): Gal2, a high affinity galactose transporter (10) that was unexpectedly found to transport glucose with nearly the same affinity (3), and Hxt2, a major glucose transporter that does not transport galactose (3, 10). These two transporters belong to the Glut transporter family, the largest known organic solute transporter family comprising more than 80 transporters found in prokaryotes through mammals (11,12). Creating chimeras between the Gal2 and Hxt2 transporters gave us an opportunity to study the galactose recognition site in Gal2 and to gain insights into the substrate recognition sites in Glut family transporters in general. To unequivocally determine the substrate recognition site, we have taken two steps. In the first step (3), three types of systematic chimeras were made using the Escherichia coli homologous recombination system. The site responsible for differentially recognizing galactose and glucose was localized to a 101-amino acid region that includes the transmembrane 10 (TM10), 1 TM11, and TM12 segments and the proximal half of the C-terminal hydrophilic tail. In the second step (4), the 101-amino acid region was subdivided into the above four regions by introducing five restriction enzyme sites into the corresponding segments of each gene without changing the amino acids encoded. By analyzing plasmids containing all the possible combinations of these segments inserted into the corresponding parts of Hxt2, we identified TM10 as the domain where galactose and glucose are differentially recognized. TM10 contains 35 amino acid residues, of which only 12 are different between Gal2 and Hxt2. Thus, it is reasonable to assume that the amino acid residue(s) essential for the substrate recognition can be found among these 12 residues. We employed a new comprehensive approach and found that 2 amino acid residues in TM10 are important for substrate recognition. EXPERIMENTAL PROCEDURESProduction of GAL2 and HXT2 Cassette Vectors-A DNA fragment containing GAL2 was cut out by PmaCI and EcoRI and ligated to SmaI and EcoRI sites in a multicloning site of pTV3, a YEp vector (3). The nucleotide sequence immediately following the initiation codon was modified from ATGGCAGTTGAG to ATGGCAGAATTC to create an EcoRI site, which changed the deduced amino acid sequence from Met-Ala-Val-...

A systematic series of chimeras between Gal2 galactose transporter and Hxt2 glucose transporter in yeast was produced to delineate the essential domain for substrate recognition. A domain of 101 amino acids close to the COOHterminus that has been previously identified as the critical substrate recognition region was further divided into four subdomains, by introducing five restriction enzyme sites at exactly corresponding locations of both genes without changing coding amino acids. When each of all possible 16 modified genes was expressed, all the gala&se transport-active chimeras were found to possess GalZderived transmembrane segment (TM) 10. Of the 35 amino acids in the TM10 region, only 12 differ between Gal2 and Hxt2, indicating that these 12 amino acids include the critical residue(s) responsible for the differential recognition of galactose and glucose in these transporters.

The type IV pili of plasmid R64 belonging to the type IVB group are required only for liquid mating. They consist of the major and minor components PilS pilin and PilV adhesin, respectively. PilS pilin is first synthesized as a 22-kDa prepilin from the pilS gene and is then processed to a 19-kDa mature pilin by PilU prepilin peptidase. In a previous genetic analysis, we identified four classes of the pilS mutants (T. Horiuchi and T. Komano, J. Bacteriol. 180:4613-4620, 1998). The products of the class I pilS mutants were not processed by prepilin peptidase; the products of the class II mutants were not secreted; in the class III mutants type IV pili with reduced activities in liquid mating were produced; and in the class IV mutants type IV pili with normal activities were produced. Here, we describe a novel class, class V, of pilS mutants. Mutations in the pilS gene at Gly-56 or Tyr-57 produced type IV pili lacking PilV adhesin, which were inactive in liquid mating. Residues 56 and 57 of PilS pilin are suggested to function as an interface of PilS-PilV interactions.