Integration of a chlorophyll-binding protein into Escherichia coli membranes in the absence of chlorophyll.
The mechanism by which a protein integrates posttranslationally into a membrane can involve the composition of the membrane itself, domains within the inserting polypeptide, and a number of associating proteins. Some integral membrane proteins do not accumulate to normal levels when certain pigments are deficient, and this has been interpreted to mean that such proteins may be rapidly degraded when not in a correct complex. Alternatively, pigments could facilitate the movement of some proteins from an aqueous to a lipid environment. To determine whether chlorophyll is absolutely required for the membrane integration of the light-harvesting chlorophyll-binding protein (LHCP) of chloroplast thylakoid membranes, we have expressed LHCP in Escherichia coli that lacks photosynthetic pigments. LHCP is targeted to the bacterial inner membrane by the addition of a bacterial signal peptide and cannot be extracted from these membranes by NaOH, NaBr, or Na2HCO3 but is extracted by 0.2% Triton X-100. Treatment of isolated right-side-out and inside-out bacterial inner membrane vesicles with trypsin reveals that only the amino terminus of LHCP is exposed on the cytoplasmic face, and the remaining portion of the protein is inaccessible. Treatment of the inside-out vesicles with trypsin followed by alkaline extraction shows that LHCP is intrinsic to the membrane and is not anchored solely by the bacterial signal peptide. Chlorophyll, therefore, is not required for LHCP to integrate into a membrane, but in the absence of these pigments this process is observed to be inefficient.
Kohorn
B D
BD
Department of Botany, Duke University, Durham, North Carolina 27706.
Auchincloss
A H
AH
D000595:Amino Acid Sequence; D001483:Base Sequence; D002473:Cell Wall / Q000378:metabolism*; D002734:Chlorophyll / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D006868:Hydrolysis; D045342:Light-Harvesting Protein Complexes; D008969:Molecular Sequence Data; D045322:Photosynthetic Reaction Center Complex Proteins / Q000378:metabolism*; D011499:Protein Processing, Post-Translational
D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; D002734:Chlorophyll