Author

Bing NaFollow

Date of Award

8-10-2007

Degree Type

Closed Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Dr. Phang C. Tai - Chair

Second Advisor

Dr. Chung-Dar

Third Advisor

Dr. Houghton John

Fourth Advisor

Dr. Kaur Parjit

Abstract

E. coli SecA is an essential component for protein translocaiton across membrane. SecA can be deleted from its N- and/or C-terminal ends without losing complementation activity. In this study, we determined the dispensity of both ends of SecA molecule. The minimal length at the SecA C-terminus is dependent on the length of the N-terminal region. SecA10-826 and SecA22-829 are the two minimal length SecAs. One more amino acid deleted at the C-terminal end completely abolished their complementation activity. A hydrophobic amino acid is required at the 826th amino acid in the minimal-length SecAs. Both SecA22-828 and SecA22-829 could form a dimer, and have decreased ATPase and protein translocation activities. The active truncated SecA mutants tended to have more soluble form than membrane-bound form, but were stably embedded in membrane. In contrast, the inactive truncated SecA mutants tended to have more membrane-bound form, but were not stable in membrane. Thus, the loss of complementation is not related to dimerization, ATPase and translocation activity but to certain extent related to their biased subcelluar localization and conformation in membrane. Isolated membranes of E coli strains were solubilized and fractionated by sucrose gradient fractionation. These membranes fractions were depleted of SecY and YidC, but contained SecD, SecF and GroEL. Proteoliposomes reconstituted from these fractionated membrane proteins were active in pOmpA translocation which required SecA and ATP. Membrane fractions from strain CK1801 in which the unc gene is deleted were reconstituted into liposomes and also showed translocation activities. Moreover, proteoliposomes reconstituted with Bacteriorodopsin alone were not active in translocation, while proteoliposomes reconstituted with Bacteriorodopsin and CK1801 membrane fractions showed elevated translocation efficiency. These data suggested that proton motive force is not obligatory for, but stimulatory to translocation of pOmA. Purified GroEL was reconstituted into lipsomes and the reconstituted proteoliposomes were active in pOmpA translocation although at lower efficiency. This translocation also required SecA and ATP. These results together suggested that translocation of pOmpA is active in the absence of SecY and YidC. In the absence of SecYEG, translocation of pOmpA requires SecA and ATP. GroEL, SecD and SecF may participate in the SecY-independent translocation.

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