Characterization of purified and unidirectionally reconstituted Pho84 phosphate permease of Saccharomyces cerevisiae.
Hydropathy analysis of the amino acid sequence of the Pho84 phosphate permease of Saccharomyces cerevisiae suggests that the protein consists of 12 transmembrane domains connected by hydrophilic loops. The Pho84 protein has been modified by a gene fusion approach, yielding two different N-terminal His-tagged chimeras which can be expressed in Escherichia coli, purified and functionally reconstituted into defined proteoliposomes. The continuous epitopes in the N- and C-terminal sequences of the Pho84 chimeras were shown to be accessible in proteoliposomes containing the purified active Pho84 proteins. Site-specific proteolysis of the immunoreactive N-terminal sequence in the reconstituted protein suggests a unidirectional insertion into liposomes.
Fristedt
U
U
Department of Biochemistry, Wallenberg Laboratory, Stockholm University, Sweden.
Weinander
R
R
Martinsson
H S
HS
Persson
B L
BL
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000493:pharmacokinetics; D002462:Cell Membrane / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D005656:Fungal Proteins / Q000737:chemistry*; D026901:Membrane Transport Proteins / Q000737:chemistry*; D008954:Models, Biological; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011510:Proteolipids / Q000378:metabolism; D028081:Proton-Phosphate Symporters; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D013997:Time Factors
D000596:Amino Acids; D002352:Carrier Proteins; D005656:Fungal Proteins; D026901:Membrane Transport Proteins; C105495:PHO84 protein, S cerevisiae; D011510:Proteolipids; D028081:Proton-Phosphate Symporters; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C020485:proteoliposomes