MEMBRANE TOPOLOGY AND MUTATIONAL ANALYSIS OF THE TOLQ PROTEIN OF ESCHERICHIA-COLI REQUIRED FOR THE UPTAKE OF MACROMOLECULES AND CELL-ENVELOPE INTEGRITY

TolQ is a 230-amino-acid protein required to maintain the integrity of the bacterial envelope and to facilitate the import of both filamento us bacteriophage and group A colicins. Cellular fractionation experime nts showed TolQ to be localized to the cytoplasmic membrane. Bacteria expressing a series of TolQ-beta-galactosidase and TolQ-alkaline phosp hatase fusion proteins were analyzed for the appropriate enzyme activi ty, membrane Location, and sensitivity to exogenously added protease. The results are consistent with TolQ being an integral cytoplasmic mem brane protein with three membrane-spanning regions. The amino-terminal 19 residues as well as a small loop in the 155 to 170 residue region appear exposed in the periplasm, while the carboxy terminus and a larg e loop after the first transmembrane region are cytoplasmic. Amino-ter minal sequence analysis of TolQ purified from the membrane revealed th e presence of the initiating formyl methionine group, suggesting a rap id translocation of the amino-terminal region across the cytoplasmic m embrane. Analysis of various tolQ mutant strains suggests that the thi rd transmembrane region as well as parts of the large cytoplasmic loop are necessary for activity.