SecY variants that interfere with Escherichia coli protein export in the presence of normal secY
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T Shimoike1, Y Akiyama, T Baba
1Department of Cell Biology, Kyoto University, Japan.
Abstract:
As an approach for studying how SecY, an integral membrane protein translocation factor of Escherichia coli, interacts with other protein molecules, we isolated a dominant negative mutation, secY-d1, of the gene carried on a plasmid. The mutant plasmid severely inhibited export of maltose-binding protein and less severely of OmpA, when introduced into sec+ cells. It inhibited growth of secY and secE mutant cells, but not of secA and secD mutant cells or wild-type cells. The mutation deletes three amino acids that should be located at the interface of cytoplasmic domain 5 and transmembrane segment 9. We also found that some SecY-PhoA fusion proteins that lacked carboxy-terminal portions of SecY but retain a region from periplasmic domain 3 to transmembrane segment 7 were inhibitory to protein export. We suggest that these SecY variants are severely defective in catalytic function of SecY, which requires cytoplasmic domain 5 and its carboxy-terminal side, but retain the ability to associate with other molecules of the protein export machinery, which requires the central portion of SecY; they probably exert the 'dominant negative' effects by competing with normal SecY for the formation of active Sec complex. These observations should provide a basis for further genetic analysis of the Sec protein complex in the membrane.
Insights
A dominant negative mutation in SecY, an integral membrane protein translocation factor in Escherichia coli, disrupts protein export. This mutation interferes with the SecY complex formation, providing insights into the protein export machinery.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- SecY is an integral membrane protein essential for protein translocation across the bacterial inner membrane in Escherichia coli.
- Understanding SecY's interactions is crucial for elucidating the mechanism of the protein export machinery.
Purpose of the Study:
- To investigate the interactions of SecY with other protein molecules.
- To characterize a dominant negative mutation (secY-d1) affecting SecY function and protein export.
Main Methods:
- Isolation and characterization of a dominant negative SecY mutation (secY-d1) using plasmid-based expression.
- Assessing the impact of the mutant SecY on the export of specific proteins (maltose-binding protein, OmpA).
- Evaluating the effect of the mutation on the growth of various Escherichia coli mutant strains (secY, secE, secA, secD).
- Construction and analysis of SecY-PhoA fusion proteins to map functional domains.
Main Results:
- The secY-d1 mutation, deleting three amino acids at a critical interface, severely inhibited protein export.
- The mutant SecY specifically affected the growth of secY and secE mutants, but not secA, secD, or wild-type cells.
- SecY-PhoA fusion proteins lacking the carboxy-terminal portion also inhibited protein export, suggesting a defect in catalytic function.
Conclusions:
- The dominant negative effect of secY-d1 is likely due to competition with normal SecY for the assembly of the active Sec complex.
- The findings suggest that distinct regions of SecY are involved in catalytic function and complex association.
- These results provide a foundation for further genetic studies of the membrane-bound Sec protein complex.