The gene encoding the phosphatidylinositol transfer protein is essential for cell growth.
Phosphatidylinositol transfer proteins (PI-TPs) catalyze the transfer of phosphatidylinositol and phosphatidylcholine between membranes in vitro. However, the in vivo function of these proteins is unknown. In this paper, we use a combined biochemical and genetic approach to determine the importance of PI-TP in vivo. An oligonucleotide based on the amino-terminal sequence of the PI-TP from Saccharomyces cerevisiae was used to screen a yeast genomic library for the gene encoding PI-TP (PIT1 gene). Positive clones showed overproduction of transfer activities and transfer protein in the 100,000 x g supernatants. The 5' terminus of the PIT1 gene correlates with the predicted codons for residues 3-30 of the determined protein sequence. A putative intron is located between the codons for residues 2 and 3 of the protein sequence. The codons for the first two amino acids of the protein and the presumptive initiation methionine precede the intron. Tetrad analysis of a heterozygous diploid (PIT1/pit1::LEU2) revealed that the PIT1 gene is essential for cell growth. Nonviable spores could be rescued by transformation of the above diploid prior to sporulation, with a plasmid-borne copy of the wild type gene.
Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston 77225.
J F
JF
Aitken
G P
GP
van Heusden
M
M
Temkin
W
W
Dowhan
D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003062:Codon; D015342:DNA Probes; D004926:Escherichia coli / Q000235:genetics; D007438:Introns; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010716:Phosphatidylinositols / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins; D010957:Plasmids; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000032:analysis / Q000235:genetics / Q000254:growth & development*; D029701:Saccharomyces cerevisiae Proteins; D013172:Spores, Fungal / Q000502:physiology; D014170:Transformation, Genetic
D002352:Carrier Proteins; D003062:Codon; D015342:DNA Probes; D008565:Membrane Proteins; D010716:Phosphatidylinositols; D049294:Phospholipid Transfer Proteins; C118289:SEC24 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins