Related Experiment Videos
Topogenesis of peroxisomal membrane protein requires a short, positively charged intervening-loop sequence and
1Department of Biology, Faculty of Sciences, Kyushu University Graduate School, Fukuoka 812-8581, Japan.
Abstract:
Human 34-kDa peroxisomal membrane protein (PMP34) consisting of 307 amino acids was previously identified as an ortholog of, or a similar protein (with 27% identity) to the, 423-amino acid-long PMP47 of the yeast Candida boidinii. We investigated membrane topogenesis of PMP34 with six putative transmembrane segments, as a model peroxisomal membrane protein. PMP34 was characterized as an integral membrane protein of peroxisomes. Transmembrane topology of PMP34 was determined by differential permeabilization and immunofluorescent staining of HeLa cells ectopically expressing PMP34 as well as of Chinese hamster ovary-K1 expressing epitope-tagged PMP34. As opposed to PMP47, PMP34 was found to expose its N- and C-terminal parts to the cytosol. Various deletion variants of PMP34 and their fusion proteins with green fluorescent protein were expressed in Chinese hamster ovary-K1 and were verified with respect to intracellular localization. The loop region between transmembrane segments 4 and 5 was required for the peroxisome-targeting activity, in which Ala substitution for basic residues abrogated the activity. Three hydrophobic transmembrane segments linked in a flanking region of the basic loop were essential for integration of PMP34 to peroxisome membranes. Therefore, it is evident that the intervening basic loop plus three transmembrane segments of PMP34 function as a peroxisomal targeting and topogenic signal.
Insights
The human peroxisomal membrane protein 34 (PMP34) integrates into peroxisome membranes. A specific loop and transmembrane segments within PMP34 act as a targeting signal for peroxisomes.
Area of Science:
- Cell Biology
- Molecular Biology
- Protein Trafficking
Background:
- Human 34-kDa peroxisomal membrane protein (PMP34) is an ortholog of yeast PMP47.
- PMP34 is an integral membrane protein with six putative transmembrane segments.
- Understanding peroxisomal membrane protein topogenesis is crucial for cellular function.
Purpose of the Study:
- To investigate the membrane topogenesis and peroxisomal targeting of human PMP34.
- To identify the specific regions of PMP34 responsible for its integration and localization to peroxisomes.
Main Methods:
- Differential permeabilization and immunofluorescent staining of cells expressing PMP34 variants.
- Expression of deletion variants and green fluorescent protein (GFP) fusion proteins in Chinese hamster ovary-K1 cells.
- Analysis of intracellular localization and membrane integration.
Main Results:
- PMP34 was confirmed as an integral peroxisomal membrane protein with N- and C-termini exposed to the cytosol.
- The loop region between transmembrane segments 4 and 5 is essential for peroxisome targeting.
- Three hydrophobic transmembrane segments flanking this loop are critical for membrane integration.
Conclusions:
- The loop region between transmembrane segments 4 and 5, along with adjacent transmembrane segments, constitutes a peroxisomal targeting and topogenic signal for PMP34.
- This signal directs PMP34 integration and localization to the peroxisomal membrane.