EFFICIENT INSERTION OF ODD-NUMBERED TRANSMEMBRANE SEGMENTS OF THE TETRACYCLINE RESISTANCE PROTEIN REQUIRES EVEN-NUMBERED SEGMENTS

Functional membrane insertion elements in the pBR322 tetracycline resi stance protein were identified by comparing the ability of odd-numbere d transmembrane segments and their attached periplasmic loops to inser t into the membrane individually or when combined with the next even-n umbered segment in the tetracycline resistance protein sequence, The e fficiency with which individual odd-numbered segments and periplasmic loops inserted was probed by treating proteins truncated at the distal ends of periplasmic loops P2-P6 with carboxypeptidases and endoprotea ses in inside-out membrane vesicles, Insertion of odd-numbered segment s and attached loops is inefficient when they occupy a C-terminal posi tion in the protein, The C-terminal odd-numbered segment and loop sequ ences of 34-54% of the molecules of periplasmic loop truncation mutant s could be removed by carboxypeptidase Y, In contrast, odd-numbered se gments and loops insert efficiently if the next even-numbered segment in the sequence is present, In such cytoplasmic loop truncation mutant s, only the cytoplasmic tail sequences of the proteins could be remove d by carboxypeptidases. Remarkably, insertion of individual odd-number ed segments and loops is inefficient even though free energies for ins ertion of these sequences are highly favorable. The results indicate t hat pairs of adjacent segments, possibly ''helical hairpins,'' are nec essary for efficient membrane insertion of the tetracycline resistance protein.