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Two different targeting signals direct human peroxisomal membrane protein 22 to peroxisomes
Ute Brosius1, Thomas Dehmel, Jutta Gärtner
1Department of Pediatrics, Heinrich Heine University, Düsseldorf D-40225, Germany.
Abstract:
The 22-kDa peroxisomal membrane protein (PMP22) is a major component of peroxisomal membranes in mammals. Although its precise role in peroxisome function is poorly understood, it seems to be involved in pore forming activity and may contribute to the unspecific permeability of the organelle membrane. PMP22 is synthesized on free cytosolic ribosomes and then directed to the peroxisome membrane by specific targeting information. Previous studies in rats revealed that PMP22 contains one distinct peroxisomal membrane targeting signal in the amino-terminal cytoplasmic tail. We cloned and characterized the targeting signal of human PMP22 and compared it with the already described characteristics of the corresponding rat protein. Amino acid sequence alignment of rat and human protein revealed 77% identity including a high conservation of several protein motifs. We expressed various deletion constructs of PMP22 in fusion with the green fluorescent protein in COS-7 cells and determined their intracellular localization. In contrast to previous studies on rat PMP22 and most other peroxisomal membrane proteins, we showed that human as well as rat PMP22 contains two distinct and nonoverlapping peroxisomal membrane targeting signals, one in the amino-terminal and the other in the carboxyl-terminal protein region. They consist of two transmembrane domains and adjacent protein loops with almost identical basic clusters. Both of these peroxisomal targeting regions interact with PEX19, a factor required for peroxisome membrane synthesis. In addition, we observed that fusing the green fluorescent protein immediately adjacent to the targeting region completely abolishes targeting function and mislocalizes PMP22 to the cytosol.
Insights
Human and rat peroxisomal membrane protein 22 (PMP22) possess two distinct targeting signals for peroxisome membranes. These signals interact with PEX19, a key factor in peroxisome biogenesis.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Peroxisomal membrane protein 22 (PMP22) is a major mammalian peroxisomal membrane component.
- Its exact function in peroxisome biology remains unclear, but it may influence membrane permeability.
- PMP22 is synthesized in the cytosol and targeted to peroxisomes via specific signals.
Purpose of the Study:
- To characterize the peroxisomal targeting signals of human PMP22.
- To compare human PMP22 targeting with previously studied rat PMP22.
- To investigate the interaction of PMP22 targeting regions with PEX19.
Main Methods:
- Cloning and characterization of human PMP22 targeting signals.
- Amino acid sequence alignment of human and rat PMP22.
- Expression of PMP22 deletion constructs fused to green fluorescent protein (GFP) in COS-7 cells.
- Determination of intracellular localization of expressed constructs.
Main Results:
- Human and rat PMP22 share 77% amino acid identity, with conserved motifs.
- Contrary to prior studies, both human and rat PMP22 possess two non-overlapping targeting signals: one N-terminal and one C-terminal.
- These signals correspond to transmembrane domains and interact with PEX19.
- Fusion of GFP directly adjacent to targeting regions abolished function, leading to cytosolic mislocalization.
Conclusions:
- Human and rat PMP22 utilize two distinct peroxisomal membrane targeting signals.
- These signals, involving transmembrane domains, interact with PEX19, crucial for peroxisome membrane synthesis.
- The precise location of targeting signals is critical for PMP22 localization to peroxisomes.