Abstract
Recent studies on the sorting of peroxisomal membrane proteins challenge the long-standing model in which peroxisomes are considered to be autonomous organelles that multiply by growth and division. Here, we present data lending support to the idea that the endoplasmic reticulum (ER) is involved in sorting of the peroxisomal membrane protein Pex3p, a protein required early in peroxisome biogenesis. First, we show that the introduction of an artificial glycosylation site into the N terminus of Pex3p leads to partial N-linked core glycosylation, indicative of insertion into the ER membrane. Second, when FLAG-tagged Pex3p is equipped with an ER targeting signal, it can restore peroxisome formation in pex3Δ cells. Importantly, FLAG antibodies that specifically recognize the processed Pex3p show that the signal peptide of the fusion protein is efficiently cleaved off and that the processed protein localizes to peroxisomes. In contrast, a Pex3p construct in which cleavage of the signal peptide is blocked by a mutation localizes to the ER and the cytosol and cannot complement pex3Δ cells. Together, these results strongly suggest that ER-targeted Pex3p indeed routes via the ER to peroxisomes, and we hypothesize that this pathway is also used by endogenous Pex3p.
Highlights
Trafficking of peroxisomal membrane proteins (PMPs)2 is less well understood, two observations indicate that distinct pathways exist for targeting of membrane and matrix proteins
PMPs do not contain a PTS1 or PTS2 but, rather, possess a distinct membrane peroxisomal targeting signal consisting of a transmembrane domain in conjunction with a cluster of basic residues that frequently contains a number of hydrophobic residues [7,8,9,10,11]
In an attempt to find evidence for routing of Pex3p through the endoplasmic reticulum (ER), we epitope-tagged full-length Pex3p at its N terminus with a synthetic NH tag (MQDLPGNDNSTAGGS), which corresponds to the N terminus of mature hemagglutinin and contains a single N-glycosylation site (NH-Pex3p, see Fig. 1 and Ref. [38]. pex3⌬ cells expressing NH-Pex3p from the strong catalase promoter were first analyzed for growth on oleate as the sole carbon source and for peroxisomal import of PTS1-tagged green fluorescent protein (GFP-SKL)
Summary
PEX3-F-207 PEX3-R-1 PEX3Eag-F PEX3-R SP-F FLAGSP-R FLAGPEX3-F FLAGSP*Y-R FLAG-F1 PEX3Myc-R. Sequence CCGGAATTCAGCTAAGATGTCGTTAACAAG GCCGAGCTCCCTTTTAGTGGTTTGCCTCC AATTATCGGCCGTTCCAAATCAAAGATCACGTTCG ACATGCATGCTTAAGGCTTGAAGGAAAACGAG CGAGCTCATCACACAAACAAAC CTTGTCATCGTCGTCCTTGTAGTCCGCAGATATTTTGGCTGCAAAAC GACTACAAGGACGACGATGACAAGGGATCCGCCCCAAATCAAAGATCACG CTTGTCATCGTCGTCCTTGTAGTCATAAGATATTTTGGCTGCAAAAC GCCGAGCTCATGGACTACAAGGACGACG ACATGCATGCTTACAAATCTTCTTCAGAAATCAATTTTTGTTCAGAACCAGAA CCCTGCAGAGGCTTGAAGGAAAACGAGC general role in PMP routing. Arguments against such a role are divers (for an in depth discussion, see Ref. 3). The processed Pex3p reaches the peroxisome and can rescue the pex phenotype of a pex3⌬ strain, whereas a construct in which cleavage of the signal peptide is blocked fails to do so. Together, these studies provide strong evidence for an ER-to-peroxisome pathway of Pex3p
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