Small proteins of less than 50 amino acids have largely been overlooked in genome annotation and are not detected by many biochemical and genetic approaches.  However, increasing evidence suggests that these proteins serve as important regulators of membrane proteins.  For example, we found that the 49-amino acid AcrZ protein associates with the AcrAB-TolC multidrug efflux pump which confers resistance to a wide variety of antibiotics and other compounds in Escherichia coli.  Co-purification of AcrZ with AcrB in the absence of both AcrA and TolC, two-hybrid assays and suppressor mutations indicate that this interaction occurs through the inner membrane protein AcrB. The highly conserved acrZ gene is coregulated with acrAB through induction by the MarA, Rob, and SoxS transcription regulators. In addition, mutants lacking AcrZ are sensitive to many, but not all, of the antibiotics transported by AcrAB-TolC. This differential antibiotic sensitivity suggests that AcrZ enhances the ability of the AcrAB-TolC pump to export certain classes of substrates.