Identification of a human PTS1 receptor docking protein directly required for peroxisomal protein import

Identification of a human PTS1 receptor docking protein directly required for peroxisomal protein import

^*Department of Biology, University of California at San Diego, La Jolla, CA 92093-0322; and ^†Departement Moleculaire Celbiologie, Katholieke Universiteit Leuven–Faculteit Geneeskunde–Campus Gasthuisberg, Afdeling Farmakologie, B-3000, Belgium

Communicated by Maarten J. Chrispeels, University of California at San Diego, La Jolla, CA (received for review March 31, 1998)

Abstract

The discovery of many fatal human disorders resulting from impaired peroxisomal protein import makes the functional characterization of human peroxins critical. As part of our attempt to identify novel human genes and gene products involved in the import of peroxisomal proteins, we raised antisera against peroxisomal membrane proteins. One such antiserum inhibited peroxisomal protein import in semipermeabilized mammalian cells. This “import inhibiting” antiserum, ab-MF3, specifically recognized a 57-kDa protein. Immunoblot analysis of rat liver subcellular fractions demonstrated that this protein was present exclusively in peroxisomal membranes. Functional analysis revealed that this 57-kDa molecule bound the PTS1 receptor, Pex5p, in ligand blots, suggesting it is a docking site on the peroxisomal membrane. Previous studies have identified two yeast proteins, Pex14p and Pex13p, as Pex5p-binding proteins. To facilitate the biochemical analysis of peroxisomal membrane docking proteins, we cloned and expressed the previously unidentified human Pex14p, as well as a human Pex13p that is 39 aa longer than previously reported. Recombinant Pex14p was specifically recognized by the “import inhibiting” ab-MF3 and bound Pex5p and the Src homology 3 (SH3) domain of Pex13p in ligand blots. These studies demonstrate that the ab-MF3-immunoreactive, 57-kDa peroxisomal membrane protein is Pex14p. Furthermore, this peroxin interacts with Pex5p and Pex13p(SH3) and is directly required for peroxisomal protein import.

Footnotes

↵ ‡ M.F. and S.R.T. contributed equally to this work.
↵ § To whom reprint requests should be addressed at: Department of Biology, Room 3230, Bonner Hall, 9500 Gilman Drive, University of California at San Diego, La Jolla, CA 92093-0322. e-mail: ssubramani{at}ucsd.edu.
Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. AF045186 and AF048755 for human PEX14 and PEX13, respectively).
ABBREVIATIONS:

PTS,

peroxisomal targeting signal;

SH3,

Src homology 3;

EST,

expressed sequence tag;

PMP,

peroxisomal membrane protein