SIGNAL SEQUENCE-DEPENDENT FUNCTION OF THE TRAM PROTEIN DURING EARLY PHASES OF PROTEIN-TRANSPORT ACROSS THE ENDOPLASMIC-RETICULUM MEMBRANE

Cotranslational translocation of proteins across the mammalian ER memb rane involves, in addition to the signal recognition particle receptor and the Sec61p complex, the translocating chain-associating membrane (TRAM) protein, the function of which is still poorly understood, Usin g reconstituted proteoliposomes, we show here that the translocation o f most, but not all, secretory proteins requires the function of TRAM. Experiments with hybrid proteins demonstrate that the structure of th e signal sequence determines whether or not TRAM is needed, Features t hat distinguish TRAM-dependent and -independent signal sequences inclu de the length of their charged, NH2-terminal region and the structure of their hydrophobic core. In cases where TRAM is required for translo cation, it is not needed for the initial interaction of the ribosome/n ascent chain complex with the ER membrane but for a subsequent step in side the membrane in which the nascent chain is inserted into the tran slocation site in a protease-resistant manner. Thus, TRAM functions in a signal sequence-dependent manner at a critical, early phase of the translocation process.