Cloning and characterization of the mitochondrial phosphate transport protein gene from the yeast Saccharomyces cerevisiae.
We have cloned the gene of the Saccharomyces cerevisiae phosphate transport protein (PTP), a member of the mitochondrial anion transport protein gene family. As PTP has a blocked N-terminus, we prepared three peptides. Oligonucleotides, based on their sequences, were used to screen a Yep24-housed genomic library. A total of 2073 bases of clone Y22 code for a 311 amino acid protein (Mr 32,814), which has similarities to the anion transport proteins: a triplicate gene structure and 6 hydrophobic segments. Typical for PTP, the triplicate gene structure possesses the X-Pro-X-(Asp/Glu)-X-X-(Lys/Arg)-X-(Arg/Lys)-X (X is an unspecified amino acid) motif and the very high homology only between the first and second repeat. The 6 hydrophobic segments harbor most of the 116 amino acids that are conserved between the yeast and the beef proteins. An N-terminal-extended signal sequence, as found in the beef protein, is absent. The yeast protein has about 33% fewer basic and acidic amino acids and five fewer Cys residues than the beef protein. The protein is insensitive to N-ethylmaleimide since Cys-42 (beef) has been replaced with a Thr. Mersalyl sensitivity has been retained and must be due to one of its three cysteines. Among these three cysteines, only Cys-28, located in the first hydrophobic segment, is conserved between the yeast and the beef protein.
Phelps
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Department of Metabolic Regulation, Boston Biomedical Research Institute, Massachusetts 02114.
Schobert
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CT
Wohlrab
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002417:Cattle; D003001:Cloning, Molecular; D003488:Cyanogen Bromide; D015723:Gene Library; D005800:Genes, Fungal; D008565:Membrane Proteins / Q000235:genetics; D008928:Mitochondria / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000378:metabolism; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010446:Peptide Fragments / Q000302:isolation & purification; D028044:Phosphate-Binding Proteins; D011487:Protein Conformation; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D002352:Carrier Proteins; D008565:Membrane Proteins; D015345:Oligonucleotide Probes; D010446:Peptide Fragments; D028044:Phosphate-Binding Proteins; D003488:Cyanogen Bromide