Considerable progress has been made recently in our understanding of proteintranslocation into endoplasmic reticulum. However, the molecular mechanisms that accomplish signal sequence dependent protein translocation across andintegration into the endoplasmic reticulum (ER) membrane has not been clarified,partly because of the huge and complicated composition of the ribosome-associated nascent chain complex and partly because each process must be involved in several different environments. Very little is now known about the environment and conformation of the signal sequence as it is synthesized by the ribosome, associated with the signal recognition particle, and incorporated into the ER membrane. We have previously investigated protein trafficking from the point of view of the signal sequence by incorporating fluorescent dyes into the ribosome-associated nascent chain as it is being synthesized by the ribosome. By examining functional, fully assembled, and intact translocation complexes with probes in the signal sequence, we have elucidated several important molecular mechanistic aspects of these processes. We now propose a project to extend our unique fluorescence and fluorescence resonance energy transfer (FRET) investigation of protein translocation by addressing questions that include: 1. What is the conformation of the signal sequence of a nascent peptide? Are they different in the ribosomal tunnel and exposed to the cytosol? Is the conformation altered as it associated with the signal recognition particle? Does it become alpha helix like conformation when it inserts into ER membrane? 2. What is the topology the signal sequence when it associates with the signal recognition particle? Is the hydrophobic segment of the signal sequence completely buried into M-domain of SRP54 proteins as it binds to SRP?
