Characterization of chloroplast transit peptides and the major stromal Hsp70, CSS1 : implications for an ATP-dependent chloroplast protein import molecular motor

Chloroplast protein import is a relatively poorly understood protein trafficking system. In other protein import systems, the translocation "machinery" has been identified and well studied, including the mechanism by which proteins are unidirectionally transported across the importing membrane. In the chloroplast, no such "molecular motor" is acknowledged. The work described in this dissertation is a preliminary attempt to assign that role to the major stromal Hsp70, CSS1. We have shown, through a variety of in vivo and in vitro techniques, interaction between a chloroplast transit peptide and two members of the Hsp70 class of molecular chaperones, DnaK and CSS1. We have also mapped this specific interaction to the N-terminus of one transit peptide and generallized this N-terminal bias to all transit peptides through statistical analyses. Futhermore, we have generated a recombinant form of CSS1 and have developed a novel chromatographic technique to purify it in an active form. Finally, we have biochemically characterized CSS1, relating its place within the Hsp70 protein family and describing its catalytic and chaperone activities in detail. This work provides the basis for further in vivo and in vitro studies which our data predict will prove that CSS1 is the chloroplast protein import molecular motor.