Characterization of purified and unidirectionally reconstituted Pho84 phosphate permease of Saccharomyces cerevisiae

Hydropathy analysis of the amino acid sequence of the Pho84 phosphate perme ase of Saccharomyces cerevisiae suggests that the protein consists of 12 tr ansmembrane domains connected by hydrophilic loops. The Pho84 protein has b een modified by a gene fusion approach, yielding two different N-terminal H is-tagged chimeras which can be expressed in Escherichia coli, purified and functionally reconstituted into defined proteoliposomes, The continuous ep itopes in the N- and C-terminal sequences of the Pho84 chimeras were shown to be accessible in proteoliposomes containing the purified active Pho84 pr oteins. Site-specific proteolysis of the immunoreactive N-terminal sequence in the reconstituted protein suggests a unidirectional insertion into lipo somes. (C) 1999 Federation of European Biochemical Societies.