Sorting of peroxisomal membrane protein PMP47 from Candida boidinii into peroxisomal membranes of Saccharomyces cerevisiae.
A gene encoding PMP47, a peroxisomal integral membrane protein of the methylotrophic yeast Candida boidinii, was isolated from a genomic library. DNA sequencing of PMP47 revealed an open reading frame of 1269 base pairs capable of encoding a protein of 46,873 Da. At least two membrane-spanning regions in the protein are predicted from the sequence. Since the 3 amino acids at the carboxyl terminus are -AKE, PMP47 lacks a typical peroxisomal sorting signal. No significant similarities in primary structure between PMP47 and known proteins were observed, including PMP70, a rat peroxisomal membrane protein whose sequence has recently been reported (Kamijo, K., Taketani, S., Yokota, S., Osumi, T., and Hashimoto, T. (1990). J. Biol. Chem. 265, 4534-4540). In order to study the import and assembly of PMP47 into peroxisomes by genetic approaches, the gene was expressed in the yeast Saccharomyces cerevisiae. When PMP47 was expressed in cells grown on oleic acid to induce peroxisomes, the protein was observed exclusively in peroxisomes as determined by marker enzyme analysis of organelle fractions. Most of the PMP47 co-purified with the endogenous peroxisomal membrane proteins on isopycnic sucrose gradients. Either in the native host or when expressed in S. cerevisiae, PMP47 was not extractable from peroxisomal membranes by sodium carbonate at pH 11, indicating an integral membrane association. These results indicate that PMP47 is competent for sorting to and assembling into peroxisomal membranes in S. cerevisiae.
McCammon
M T
MT
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235.
Dowds
C A
CA
Orth
K
K
Moomaw
C R
CR
Slaughter
C A
CA
Goodman
J M
JM
D000595:Amino Acid Sequence; D001483:Base Sequence; D002175:Candida / Q000235:genetics* / Q000378:metabolism; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D005656:Fungal Proteins / Q000235:genetics*; D005800:Genes, Fungal; D007425:Intracellular Membranes / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics*; D008830:Microbodies / Q000378:metabolism*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D011487:Protein Conformation; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism
D004271:DNA, Fungal; D005656:Fungal Proteins; D008565:Membrane Proteins; D015345:Oligonucleotide Probes; C066456:PMP47 protein, Candida boidinii