The Membrane Biogenesis Peroxin Pex16p: TOPOGENESIS AND FUNCTIONAL ROLES IN PEROXISOMAL MEMBRANE ASSEMBLY

Masanori Honsho,Takanobu Hiroshige,Yukio Fujiki

doi:10.1074/jbc.m206139200

Masanori Honsho, Takanobu Hiroshige + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m206139200

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Abstract

Previously we isolated human PEX16 encoding 336-amino acid-long peroxin Pex16p and showed that its dysfunction was responsible for Zellweger syndrome of complementation group D (group 9). Here we have determined the membrane topology of Pex16p by differential permeabilization method: both N- and C-terminal parts are exposed to the cytosol. In the search for Pex16p topogenic sequence, basic amino acids clustered sequence, RKELRKKLPVSLSQQK, at positions 66-81 and the first transmembrane segment locating far downstream, nearly by 40 amino acids, of this basic region were defined to be essential for integration into peroxisome membranes. Localization to peroxisomes of membrane proteins such as Pex14p, Pex13p, and PMP70 was interfered with in CHO-K1 cells by a higher level expression of the pex16 patient-derived dysfunctional but topogenically active Pex16pR176ter comprising resides 1-176 or of the C-terminal cytoplasmic part starting from residues at 244 to the C terminus. Furthermore, Pex16p C-terminal cytoplasmic part severely abrogated peroxisome restoration in pex mutants such as matrix protein import-defective pex12 and membrane assembly impaired pex3 by respective PEX12 and PEX3 expression, whereas the N-terminal cytosolic region did not affect restoration. These results imply that Pex16p functions in peroxisome membrane assembly, more likely upstream of Pex3p.

Highlights

We isolated human PEX16 encoding 336amino acid-long peroxin Pex16p and showed that its dysfunction was responsible for Zellweger syndrome of complementation group D
Membrane Topology of Pex16p—We determined the transmembrane topology of human Pex16p in peroxisomal membranes by the differential cell-permeabilization method, using CHO-K1 cells transfected with Flag-PEX16-HA encoding Nand C-terminally epitope-tagged Pex16p, where detection of Pex16p was done using antibodies to epitope tags
When the CHO-K1 cells expressing FLAG-Pex16p-HA were treated with 1% Triton X-100 which solubilized all cellular membranes, both FLAGPex16p-HA and peroxisomal targeting signal type 1 (PTS1) proteins were detected in particulates, in a superimposable manner (Fig. 1, a and c), thereby indicating localization of Pex16p in peroxisomes

Summary

Introduction

We isolated human PEX16 encoding 336amino acid-long peroxin Pex16p and showed that its dysfunction was responsible for Zellweger syndrome of complementation group D (group 9). Pex16p C-terminal cytoplasmic part severely abrogated peroxisome restoration in pex mutants such as matrix protein import-defective pex and membrane assembly impaired pex by respective PEX12 and PEX3 expression, whereas the N-terminal cytosolic region did not affect restoration. These results imply that Pex16p functions in peroxisome membrane assembly, more likely upstream of Pex3p. The sequence relatively enriched in positively charged amino acid residues and transmembrane segment(s) is mostly required for translocation of integral membrane proteins to peroxisomes, any consensus sequence for mPTS has not been delineated. The cytoplasmically oriented C-terminal part of Pex16p was likely to play a role in an early stage of peroxisome assembly

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