Our long term objectives are to understand the molecular mechanisms by which secretory proteins are targeted to, and translocated across, the appropriate cytoplasmic membrane in both eukaryotes and prokaryotes. Our general strategy is to concentrate on a single protein, the maltose- binding protein (MBP) from Escherichia coli. The mechanism(s) by which MBP is exported from E. coli is well-characterized. Detailed comparisons of the translocation of this protein in homologous bacterial systems and in heterologous eukaryotic systems will reveal similarities and differences in the mechanisms for MBP translocation in the two systems. Moreover, when expressed in eukaryotic systems, MBP will serve as a probe for novel mechanisms of protein translocation. We propose three specific aims to probe the mechanisms of MBP translocation in prokaryotes and eukaryotes: 1. Characterize the translocation of MBP across the mammalian ER membrane. MBP can be translocated across mammalian microsomes by a potentially novel post-translational mechanism. We will further characterize the translocation of MBP in mammalian cell-free systems using biochemical fractionation and reconstitution to define intramolecular interactions between MBP and accessory proteins in the cytosol and the membrane. 2. Determine steps at which translocation is blocked by specific signal sequence mutants. We will characterize eukaryotic translocation of signal sequence mutants of MBP to compare signal sequence function in bacteria and eukaryotes. Selected mutants are known to be incompletely translocated across the bacterial inner membrane and represent a series of translocation intermediates. 3. Identify cytosolic and membrane proteins which interact with MBP at different stages in its translocation across the ER and bacterial inner membranes. Chemical cross-linking of wild-type and mutant MBP will be employed to dissect out molecular components which facilitate different steps in MBP targeting and translocation. Molecular and biochemical approaches will be used to characterize cross-linking molecules.
