Pex3p‐dependent peroxisomal biogenesis initiates in the endoplasmic reticulum of human fibroblasts

Abstract

The mechanisms of peroxisomal biogenesis remain incompletely understood, specially regarding the role of the endoplasmic reticulum (ER) in human cells, where genetic disorders of peroxisome biogenesis lead to Zellweger syndrome (ZS). The Pex3p peroxisomal membrane protein (PMP) required for early steps of peroxisome biogenesis has been detected in the ER in yeast but not in mammalian cells. Here, we show that Pex3p-GFP expressed in a new ZS cell line (MR), which lacks peroxisomes due to a mutation in the PEX3 gene, localizes first in the ER and subsequently in newly formed peroxisomes. Pex3p bearing an artificial N-glycosylation site shows an electrophoretic shift indicative of ER targeting while en route to preformed peroxisomes in normal fibroblast. A signal peptide that forces its entry into the ER does not eliminate its capability to drive peroxisome biogenesis in ZS cells. Thus, Pex3p is able to drive peroxisome biogenesis from the ER and its ER pathway is not privative of ZS cells. Cross-expression experiments of Pex3p in GM623 cells lacking Pex16p or Pex16p in MR cells lacking Pex3p, showed evidence that Pex3p requires Pex16p for ER location but is dispensable for the ER location of Pex16p. These results indicate that Pex3p follows the ER-to-peroxisomal route in mammalian cells and provides new clues to understand its function.

Peroxisomal Biogenesis: Genetic Disorders Reveal the Mechanisms

“…First, it was shown that Pex16p is addressed to the ER before its sorting to peroxisomes (30). Afterwards, a similar route was revealed for Pex3p in ZS fibroblasts (26), thus providing the elusive evidence of previous studies. In wild type mammalian cells (Cos7 cells), experiments with a photoactivable Pex16p-GFP revealed a trafficking pathway initiated at the ER and leading to peroxisomes (30).…”

“…This phenotype is reproduced in yeast by PEX3 and PEX19 mutations (13,27). Strikingly, the expression of exogenous wild type PEX genes in ZS cells and mutant yeasts reestablish the generation of functional peroxisomes (13,14,19,22,(26)(27)(28)(29)(30)(31), demonstrating that new peroxisomes can be generated without requiring a preexisting organelle. These observations also indicate that early stages of peroxisome biogenesis are driven by peroxins Pex3p, Pex16p and Pex19p, respectively encoded by PEX3, PEX16 and PEX19 genes (18).…”

“…To date 32 PEX genes encoding the peroxisomal biogenetic machinery have been identified and at least 12 different complementation groups have been described among ZS patients, most of them displaying peroxisomal ghosts (7,8,18,20). However, three of these complementation groups, groups 9 (PEX16 gene defect), 12 (PEX3 gene defect) and 14 (PEX19 gene defect) lack peroxisomes, peroxisome ghosts and any peroxisomal membrane (5,7,14,(19)(20)(21)(22)(23)(24)(25)(26). This phenotype is reproduced in yeast by PEX3 and PEX19 mutations (13,27).…”

“…Plants also contributed with evidence of an ER-to-peroxisome pathway (41)(42)(43). Although in mammalian cells such possibility has been more controversial, accumulated evidence (26,30) prompts reconsidering its validity.…”

“…The evidence suggested that most peroxisomes derive from the ER pathway rather than from preexisting organelles. Prompted by the refreshing results on Pex16p traffic in living mammalian cells (30) and the contrasting observations regarding Pex3p trafficking in yeast (14,22,31) and mammalian cells (28,34), we decided to study the sorting behavior of Pex3p and Pex16p in a fibroblast cell line (called MR) derived from a Chilean patient with ZS (26). In this new MR cell line we found complete lack of peroxisomes, including peroxisomal membrane ghosts, due to nonsense mutation in the PEX3 gene.…”