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. Author manuscript; available in PMC: 2017 Oct 11.
Published in final edited form as: Biochemistry. 2016 Sep 30;55(40):5649–5652. doi: 10.1021/acs.biochem.6b00920

Figure 1.

Figure 1

An ssrA-tagged model membrane protein is degraded in E. coli. (A) Topology of ProW1-182-FLAG-ssrA (top) and amino-acid sequences of wild-type and mutant ssrA tags (bottom). (B) Expression of ProW1-182-FLAG-ssrA or ProW1-182-FLAG-ssrA(DD) in E. coli X90 was induced, pulsed with 35S-labeled methionine and cysteine, and chased with unlabeled amino acids. Samples taken at different times were immunoprecipitated and analyzed by SDS-PAGE and autoradiography. “Un” indicates an uninduced control sample. (C) E. coli cells expressing ProW1-182-FLAG-ssrA(DD) were lysed and fractionated into soluble (S) and membrane (M) fractions, which were analyzed by SDS-PAGE and immunoblotted using antibodies against GroEL (a cytosolic protein) and the FLAG tag. (D) Activity (in Miller units) of C-terminal FLAG-ssrA-PhoA fusions of a cytosolic protein (λ repressor), a periplasmic protein (cytochrome b562), and ProW1-182.