Discrete targeting signals direct Pmp47 to oleate-induced peroxisomes in Saccharomyces cerevisiae.
Pmp47 is a peroxisomal membrane protein consisting of six transmembrane domains (TMDs). We previously showed that the second matrix loop containing a basic cluster of amino acids is important for peroxisomal targeting, and similar basic targeting motifs have been found in other peroxisomal membrane proteins. However, this basic cluster by itself targets to peroxisomes very poorly. We have developed a sensitive quantitative localization assay based on the targeting of Pmp47-GFP fusion proteins to identify the important elements of the basic cluster and to search for other targeting information on Pmp47. Our data suggest that side-chain structure and position as well as charge are important for targeting by the basic cluster. Analysis of other regions of Pmp47 indicates that all TMDs except TMD2 can be eliminated or substituted without significant loss of targeting. TMD2 plus an adjacent cytoplasmic-oriented sequence is crucial for targeting. Cytoplasmic-oriented sequences from two other peroxisomal membrane proteins, ScPex15p and ScPmp22, could partially substitute for the analogous sequence in Pmp47. Targeting with high fidelity to oleate-induced peroxisomes required the following elements: the cytoplasmic-oriented sequence and TMD2, a short matrix loop containing a basic cluster, and a membrane-anchoring TMD.
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9041, USA.
X
X
Wang
M J
MJ
Unruh
J M
JM
Goodman
D000595:Amino Acid Sequence; D005656:Fungal Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D019301:Oleic Acid; D020675:Peroxisomes / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
D005656:Fungal Proteins; D008565:Membrane Proteins; C066456:PMP47 protein, Candida boidinii; D011993:Recombinant Fusion Proteins; D019301:Oleic Acid