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Structural and Functional Analysis of Pex19 in Pichia pastoris /

  • Author(s): Shang, Helen Hao
  • et al.
Abstract

The study of peroxisome biogenesis seeks to understand the underlying processes involved in the initial formation, maturation, and proliferation of peroxisomes. The two peroxins, Pex3 and Pex19, have essential functions in peroxisome biogenesis; cells lacking Pex3 or Pex19 have no detectable mature peroxisomes. Several studies have highlighted the importance of the endoplasmic reticulum (ER) in the de novo synthesis of peroxisomes, contrary to previous growth and division models. More recently, the novel Pex protein, Pex25, has also been implicated as a requirement for the de novo pathway. Nevertheless, the mechanism by which these proteins promote the de novo process is currently unknown. In this study, we have characterized several Pex19 N-and C-terminal deletions to determine the structural domains of the peroxin required for proper peroxisome membrane protein (PMP) and peroxisome assembly. Deletions in the N-terminal Pex3 binding site weakened Pex19's interactions with Pex3 but preserved interactions with Pex12, a mPTS-containing PMP known to bind with the C-terminal end of Pex19. For these constructs only, Pex25 was essential for de novo peroxisome biogenesis. In contrast, deletions in the C- terminal mPTS-binding domain of Pex19 weakened the interaction between Pex19 and Pex12, while leaving Pex19- Pex3 interactions intact. Surprisingly, import competent peroxisomes were formed in both sets of deletions although a delay of 12-18 hours in peroxisome biogenesis was observed. This delay was decreased upon the overexpression of Pex25 for both N-and C-terminal deletion mutants. Co- immunoprecipitation studies revealed that Pex25 promotes peroxisome biogenesis through strengthening Pex19 and PMP interactions, which are required for the exit of pre- peroxisomal vesicular carriers. In conclusion, the physical segregation of Pex19's Pex3 and PMP binding domains has provided novel insights into the role of Pex19 in peroxisome biogenesis, via the interaction of a central domain in Pex19 with Pex25

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