SITE-SPECIFIC PHOTOCROSS-LINKING REVEALS THAT SEC61P AND TRAM CONTACTDIFFERENT REGIONS OF A MEMBRANE-INSERTED SIGNAL SEQUENCE

A chemically charged amber suppressor TRNA was used to introduce the p hotoactivatable amino acid (Tmd)Phe at a selected position within the signal sequence of the secretory protein preprolactin. This allowed th e interactions of the NH2-terminal, the central, and the COOH-terminal regions of the signal Sequence to be investigated during insertion in to the membrane of the endoplasmic reticulum (ER). We found that diffe rent regions of the nascent chains were photocross-linked to different ER proteins. The TRAM protein (translocating chain-associating membra ne protein) contacts the NH2-terminal region of the signal sequence wh ile the mammalian Sec61p contacts the hydrophobic core of the signal s equence and regions COOH-terminal of this. These results suggest that the ER translocation complex is composed of heterologous protein subun its which contact distinct regions of nascent polypeptides during thei r membrane insertion.