Characterization of the minimal length of functional SecA in Escherichia coli
| Autor: | Hsiuchin Yang, Bing Na, Zhipeng You, Phang C. Tai |
|---|---|
| Rok vydání: | 2015 |
| Předmět: |
ATPase
Mutant Biophysics Chromosomal translocation medicine.disease_cause environment and public health Biochemistry Article Cytosol Bacterial Proteins Escherichia coli medicine Molecular Biology Adenosine Triphosphatases chemistry.chemical_classification SecA Proteins biology Escherichia coli Proteins Cell Membrane Genetic Complementation Test Membrane Transport Proteins Cell Biology Subcellular localization Molecular biology In vitro Protein Structure Tertiary Amino acid Complementation Protein Transport chemistry Mutation biology.protein bacteria Dimerization Hydrophobic and Hydrophilic Interactions Gene Deletion SEC Translocation Channels Plasmids Protein Binding |
| Zdroj: | Biochemical and Biophysical Research Communications. 456:213-218 |
| ISSN: | 0006-291X |
| DOI: | 10.1016/j.bbrc.2014.11.061 |
| Popis: | Previous studies showed that certain regions of E. coli SecA can be deleted from its N- and/or C-termini to complement a SecA amber ts mutant. In this study, we determined and characterized the dispensability of both ends of SecA molecules. With N-terminal intact or 9-aa deleted, 826aa (SecA1-826 and SecA10-826, respectively) is the minimum for complementation activity, while with N-terminus deleted by 2-21aa, SecA22-829 is the minimum. Further deletion at the C-terminus of SecA1-826/SecA10-826/SecA22-829 abolished the complementation activity in the cells. A hydrophobic amino acid is required for the 826th residue in the minimal-length SecAs. Chemical crosslinking and gel filtration result showed that both purified SecA22-828 and SecA22-829 could form a dimer. Moreover, the in vitro ATPase and protein translocation activities of SecA22-828 and SecA22-829 were similar, though lower than wild-type SecA. The active mutants had more truncated SecA in soluble than membrane-bound form, but was more stably embedded in membranes. In contrast, the inactive mutants tended to have truncated SecA more membrane-bound than soluble form, and were more loosely bound and easily chased out. Thus, the loss of complementation appears to be related to their altered subcellular localization and stability in the membranes. This study defines the substantial regions of N- and C-termini of SecA that may be deleted without losing complementation activity. |
| Databáze: | OpenAIRE |
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