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Membrane topography and topogenesis of prenylated Rab acceptor (PRA1)
1Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA. jialing-lin@ouhsc.edu
Abstract:
The mouse prenylated Rab acceptor (mPRA1) is associated with the Golgi membrane at steady state and interacts with Rab proteins. It contains two internal hydrophobic domains (34 residues each) that have enough residues to form four transmembrane (TM) segments. In this study, we have determined the membrane topography of mPRA1 in both intact cells and isolated microsomes. The putative TM segments of mPRA1 were used to substitute for a known TM segment of a model membrane protein to determine whether the mPRA1 segments integrate into the membrane. Furthermore, N-linked glycosylation scanning methods were used to distinguish luminal domains from cytoplasmic domains of mPRA1. The data demonstrate that mPRA1 is a polytopic membrane protein containing four TM segments. These TM segments act cooperatively during the translocation and integration at the endoplasmic reticulum membrane. All hydrophilic domains are in the cytoplasm, including the N-terminal domain, the linker domain between the two hydrophobic domains, and the C-terminal domain. As a result, the bulk of mPRA1 is located in the cytoplasm, supporting its postulated role in regulating Rab membrane targeting and intracellular trafficking.
Insights
Mouse prenylated Rab acceptor 1 (mPRA1) is a polytopic membrane protein with four transmembrane segments. All hydrophilic domains face the cytoplasm, supporting its role in Rab protein regulation and intracellular trafficking.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Mouse prenylated Rab acceptor 1 (mPRA1) is a Golgi-associated protein that interacts with Rab proteins.
- mPRA1 possesses two hydrophobic domains, each 34 residues long, suggesting the potential for multiple transmembrane segments.
Purpose of the Study:
- To determine the membrane topography of mPRA1 in intact cells and isolated microsomes.
- To elucidate the integration mechanism of mPRA1's transmembrane segments into the endoplasmic reticulum membrane.
- To identify the localization of mPRA1's hydrophilic domains (N-terminal, linker, C-terminal).
Main Methods:
- Membrane protein topology analysis using N-linked glycosylation scanning.
- Functional substitution assays with a model membrane protein to assess transmembrane segment integration.
- Analysis of mPRA1 in both intact cells and isolated microsomes.
Main Results:
- mPRA1 is confirmed as a polytopic membrane protein with four transmembrane segments.
- These transmembrane segments cooperate during translocation and integration into the endoplasmic reticulum membrane.
- All hydrophilic domains of mPRA1, including N-terminal, linker, and C-terminal regions, are located in the cytoplasm.
Conclusions:
- The cytoplasmic localization of mPRA1's hydrophilic domains supports its proposed function in regulating Rab protein targeting.
- mPRA1's structure facilitates its role in intracellular trafficking pathways.
- The cooperative action of transmembrane segments is crucial for mPRA1's proper membrane insertion and function.