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Endogenous SecA catalyzes preprotein translocation at SecYEG
J Eichler1, K Rinard, W Wickner
1Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755-3844, USA.
The Journal of Biological Chemistry
|August 15, 1998
Summary
SecA protein facilitates preprotein translocation in Escherichia coli exclusively through cycling at the SecYEG complex. Azide treatment specifically inhibits SecA at SecYEG, confirming its essential role in this process.
Area of Science:
- Cellular Biology
- Protein Transport
- Molecular Mechanisms
Background:
- SecA is a crucial ATPase motor protein involved in protein transport across the bacterial plasma membrane.
- SecA interacts with the SecYEG translocon complex and can also associate with lipids.
Purpose of the Study:
- To elucidate the specific role and membrane association states of SecA in preprotein translocation.
- To differentiate the functional relevance of SecA's various membrane-bound forms.
Main Methods:
- Utilized azide as a specific inhibitor of SecA deinsertion.
- Employed sucrose gradient ultracentrifugation to isolate endogenous SecA.
- Performed proteolytic fragmentation and urea extraction assays.
- Investigated translocation activity in vitro and in vivo.
Main Results:
- Azide selectively inhibits SecA cycling at SecYEG, not lipid-associated SecA.
- Endogenous and added SecA associated with SecYEG support translocation similarly.
- Proteolytic fragmentation patterns distinguish SecA cycling at SecYEG from other associations.
- SecA cycling at SecYEG is essential and sensitive to azide and anti-SecY antibodies.
Conclusions:
- SecA functions in preprotein translocation solely through its dynamic cycling at the SecYEG complex.
- Lipid-bound SecA and SecA inserted into the membrane are not directly involved in translocation initiation.