AN INTERNAL REGION OF THE PEROXISOMAL MEMBRANE-PROTEIN PMP47 IS ESSENTIAL FOR SORTING TO PEROXISOMES

Targeting sequences on peroxisomal membrane proteins have not yet been identified. We have attempted to find such a sequence within PMP47, a protein of the methylotrophic yeast, Candida boidinii. This protein o f 423 amino acids shows sequence similarity with proteins in the famil y of mitochondrial carrier proteins. As such, it is predicted to have six membrane-spanning domains. Protease susceptibility experiments are consistent with a six-membrane-spanning model for PMP47, although the topology for the peroxisomal protein is inverted compared with the mi tochondrial carrier proteins. PMP47 contains two potential peroxisomal targeting sequences (PTS1), an internal SKL (residues 320-322) and a carboxy terminal AKE (residues 421-423). Using a heterologous in vivo sorting system, we show that efficient sorting occurs in the absence o f both sequences. Analysis of PMP47-dihydrofolate reductase (DHFR) fus ion proteins revealed that amino acids 1-199 of PMP47, which contain t he first three putative membrane spans, do not contain the necessary t argeting information, whereas a fusion with amino acids 1-267, which c ontains five spans, is fully competent for sorting to peroxisomes. Sim ilarly, a DHFR fusion construct containing residues 268-423 did not ta rget to peroxisomes while residues 203-420 appeared to sort to that or ganelle, albeit at lower efficiency than the 1-267 construct. However, DHFR constructs containing only amino acids 185-267 or 203-267 of PMP 47 were not found to be associated with peroxisomes. We conclude that amino acids 199-267 are necessary for peroxisomal targeting, although additional sequences may be required for efficient sorting to, or rete ntion by, the organelles.