The assembly of integral membrane proteins is determined by features of these proteins and the protein translocation apparatus. We used alkaline phosphatase fusions to the membrane protein MalF to investigate the role of the protein translocation machinery in the arrangement of proteins in the cytoplasmic membrane of Escherichia coli. In particular, we studied the effects of prlA mutations on membrane protein topology. These mutations lie in the secY gene, which encodes a core component of the protein translocation apparatus. We find that the topology of some of the fusion proteins is changed and, in one case, is completely inverted in prlA mutants. We discuss the mechanism of prlA-mediated export and the role of the protein translocation apparatus in contributing to membrane protein topology.