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The membrane biogenesis peroxin Pex16p. Topogenesis and functional roles in peroxisomal membrane assembly
Masanori Honsho1, Takanobu Hiroshige, Yukio Fujiki
1Department of Biology, Faculty of Sciences, Kyushu University Graduate School, Fukuoka 812-8581, Japan.
Insights
Peroxin Pex16p is crucial for peroxisome membrane assembly and its dysfunction causes Zellweger syndrome. Its N- and C-termini are cytosolic, with specific regions essential for membrane integration.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Human PEX16 encodes peroxin Pex16p, a protein whose dysfunction causes Zellweger syndrome (complementation group D).
- Zellweger syndrome is a severe peroxisome biogenesis disorder.
Purpose of the Study:
- To determine the membrane topology of human Pex16p.
- To identify the topogenic sequences responsible for Pex16p integration into peroxisome membranes.
- To investigate the role of Pex16p in peroxisome assembly and the function of its different domains.
Main Methods:
- Differential permeabilization to ascertain N- and C-terminal exposure.
- Analysis of PEX16 mutations and expression of truncated forms in CHO-K1 cells.
- Assessing the impact of Pex16p variants on peroxisome restoration in pex mutants.
Main Results:
- Pex16p exhibits a topology with both N- and C-termini exposed to the cytosol.
- A basic amino acid cluster (residues 66-81) and a downstream transmembrane segment are essential for membrane integration.
- Expression of truncated Pex16p (Pex16pR176ter or C-terminal part) interfered with peroxisome protein localization and impaired peroxisome restoration in pex mutants.
Conclusions:
- Pex16p plays a vital role in peroxisome membrane assembly, likely acting upstream of Pex3p.
- The C-terminal cytoplasmic region of Pex16p is critical for its function in peroxisome biogenesis.
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.