Poster: Organelle Biogenesis

Abs # 1161: AtPex3p is targeted in suspension-cultured cells directly to peroxisomes via an N-terminal membrane targeting signal

Presenter:	Hunt, Joanne E, joanne.hunt@asu.edu
Authors	Hunt, Joanne E (A)   Trelease, Richard N (A)
Affiliations:	(A): Department of Plant Biology, Arizona State University

In yeast and mammals, the peroxin Pex3p presumably participates in the acquisition of other peroxisomal membrane proteins. Two putative Arabidopsis homologs of PEX3 (At3g18160 (form 1) and At1g48460 (form (2)) were identified. AtPex3p (form 2) is 24% identical (amino acids) to HsPex3p both 42kDa (predicted). N terminal sequences of Pex3p are highly conserved, consisting of a basic cluster of amino acids juxtaposed to a transmembrane domain (TMD). The N-terminal amino acids of Hansenula and human Pex3p are sufficient to target proteins to peroxisomes. We determined the subcellular localization and targeting signal of AtPex3p. Myc-AtPex3p, transiently expressed (5 h) in Arabidopsis and BY2 cultured cells, colocalized (immunofluorescence microscopy) with endogenous peroxisomal catalase in both cell types. To determine the sufficiency of peroxisomal targeting, constructs consisting of 40, 16, or 8 N-terminal amino acids of AtPex3p were appended to the N terminus of chloramphenicol acetyltransferase (CAT), and introduced biolistically into cells. The N-terminal 40 amino acids of AtPex3p were sufficient to target CAT to peroxisomes in both cell types. In Arabidopsis cells, the 16 and 8 N-terminal amino acids of AtPex3p localized CAT to the cytosol. In BY2 cells, 16 N-terminal amino acids of AtPex3p mislocalized CAT to a nonperoxisomal compartment, while 8 N-terminal amino acids of AtPex3p localized CAT to the cytosol. In summary, AtPex3p sorts from its site of synthesis in the cytosol directly to peroxisomes, where it likely functions as a peroxin. Its N-terminal 40 amino acids constitute a sufficient membrane peroxisomal targeting signal comprised of a cluster of six basic residues adjacent to the N-terminal-most TMD (22 amino acids). Supported by NSF grant MCB-0091826.