Topology analysis of the colicin V export protein CvaA in Escherichia coli

Abstract

The antibacterial protein toxin colicin V is secreted from Escherichia coli cells by a dedicated export system that is a member of the multicomponent ATP-binding cassette (ABC) transporter family. At least three proteins, CvaA, CvaB, and TolC, are required for secretion via this signal sequence-independent pathway. In this study, the subcellular location and transmembrane organization of membrane fusion protein CvaA were investigated. First, a series of CvaA-alkaline phosphatase (AP) protein fusions was constructed. Inner and outer membrane fractionations of cells bearing these fusions indicated that CvaA is inner membrane associated. To localize the fusion junctions, the relative activities of the fusion proteins, i.e., the amounts of phosphatase activity normalized to the rate of synthesis of each protein, as well as the stability of each fusion, were determined. These results indicated that all of the fusion junctions occur on the same side of the inner membrane. In addition, the relative activities were compared with that of native AP, and the protease accessibility of the AP moieties in spheroplasts and whole cells was analyzed. The results of these experiments suggested that the fusion junctions occur within periplasmic regions of CvaA. We conclude that CvaA is an inner membrane protein with a single transmembrane domain near its N terminus; the large C-terminal region extends into the periplasm. This study demonstrates the application of AP fusion analysis to elucidate the topology of a membrane-associated protein having only a single transmembrane domain.

Characterization of in-frame proteins encoded by cvaA, an essential gene in the colicin V secretion system: CvaA* stabilizes CvaA to enhance secretion

“…On the other hand, CvaA* is encoded in frame with CvaA and is therefore identical to CvaA except that it lacks the hydrophobic N-terminal residues. CvaA* is therefore proposed to be a cytosolic protein (8,16,36). Using in vivo labeling experiments followed by fractionation of cells producing CvaA and CvaA*, we have confirmed that CvaA resides in the inner membrane whereas CvaA* is located in the cytosol (data not shown).…”

“…Instead, this study shows that CvaA* stabilizes CvaA and this leads to increased ColV secretion. The C terminus of CvaA is predicted to reside in the periplasm (36). Therefore, a proposed stabilizing interaction of CvaA* with CvaA would probably not occur in the C terminus.…”

New functions for the three subunits of the CzcCBA cation-proton antiporter

“…Therefore, CzcC needs CzcB for its action, and it must handle the cations in cooperation with CzcB* or after the proposed CzcB*-funnelling process. CzcC might open up an escape route for the cations through the outer membrane; since CzcC is not a very hydrophobic protein (25), it might contact a hypothetical outer membrane protein, OmpY, as has been demonstrated for another transport process which involves a membrane fusion protein (34). Therefore, the zinc, cobalt, and cadmium cations might be transported across the cytoplasmic membrane by CzcA, through the periplasm by CzcB*, and across the outer membrane with the help of CzcC.…”

Membrane Topology and Insertion of Membrane Proteins: Search for Topogenic Signals

“…This systematic behavior can be used as an additional method to discriminate between fusions with a periplasmic or cytoplasmic PhoA moiety, but it is important that it is not used to normalize the activities. To demonstrate the synthesis of the fusion proteins, pulse and/or pulse-chase experiments are generally used (26,45,151,152,168,170,206,208).…”