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Distant downstream sequence determinants can control N-tail translocation during protein insertion into the
1Department of Biochemistry, Stockholm University, S-10691 Stockholm, Sweden.
The Journal of Biological Chemistry
|February 29, 2000
Summary
The number of transmembrane segments in Escherichia coli ProW protein influences its membrane insertion and N-tail translocation into the endoplasmic reticulum. At least four segments are needed for proper N-tail translocation.
Area of Science:
- Molecular Biology
- Protein Biochemistry
- Membrane Biology
Background:
- Understanding the mechanisms of membrane protein insertion and topology determination is crucial in cell biology.
- Escherichia coli inner membrane protein ProW features seven transmembrane segments and a large N-terminal tail.
Purpose of the Study:
- To investigate the membrane insertion of ProW into endoplasmic reticulum (ER)-derived microsomes.
- To determine the role of transmembrane segments in N-tail translocation and overall protein topology.
Main Methods:
- Studied membrane insertion of ProW constructs with varying numbers of transmembrane segments.
- Utilized ER-derived dog pancreas microsomes for in vitro translocation assays.
- Analyzed protein topology and N-tail translocation efficiency.
Main Results:
- Significant N-tail translocation required a minimum of four transmembrane segments.
- Fewer segments resulted in non-translocated N-tails and inverted topologies.
- N-tail translocation was promoted by N-tail shortening and positive charges after the first transmembrane segment.
Conclusions:
- At least four consecutive transmembrane segments collectively dictate ER membrane protein topology.
- N-tail size and charge influence the effects of downstream sequence determinants.
- ProW N-tail translocation across the ER membrane occurs in a C-to-N-terminal direction.