Tyr-326 plays a critical role in controlling SecA-preprotein interaction.

SecA is an essential ATP-dependent motor protein that interacts with the preprotein and translocon to drive protein translocation across the eubacterial plasma membrane. A region containing residues 267-340 has been proposed to comprise the preprotein binding site of Escherichia coli SecA. To elucidate the function of this region further, we isolated mutants using a combination of region-specific polymerase chain reaction (PCR) mutagenesis and a genetic and biochemical screening procedure. Although this region displayed considerable plasticity based on phylogenetic and genetic analysis, Tyr-326 was found to be critical for SecA function. secA mutants with non-conservative substitutions at Tyr-326 showed strong protein secretion defects in vivo and were completely defective for SecA-dependent translocation ATPase activity in vitro. The SecA-Y326 mutant proteins were normal in their membrane, SecYE and nucleotide-binding properties. However, they exhibited a reduced affinity for preprotein and were defective in preprotein release, as assessed by several biochemical assays. Our results indicate that the region containing Tyr-326 functions as a conformational response element to regulate the preprotein binding and release cycle of SecA.

The early stage of chloroplast protein import involves Com70.

The chloroplast envelope protein Com70 is a hsp70 homolog identified recently as a component of the protein translocation apparatus. The stage of protein import involving Com70 was determined by examining the nature of the association of Com70 with the envelope and its interaction with translocating proteins. Com70 is accessible to thermolysin, but its association with the envelope could not be disrupted by stringent washes. In light of the external membrane-bound location, the involvement of Com70 at the early stage of protein translocation was investigated using a combination of in vitro binding assays, chemical cross-linking, and coimmunoprecipitation. The results provide evidence that Com70 is in close physical proximity to different types of chloroplast protein precursors under conditions supporting binding rather than complete translocation. The formation of cross-linked complexes is dependent on the presence of a typical plastid transit signal and protease-accessible outer envelope components. The close proximity of Com70 and the translocating protein occurs while the protein is still exposed to the cytosol.

Isolation and characterization of a cDNA clone encoding a member of the Com44/Cim44 envelope components of the chloroplast protein import apparatus.

Many of the proteins in the chloroplast envelope play an important role in facilitating the biochemical and transport processes of the compartment. For the transport of proteins into the chloroplast, we have recently identified at least three different envelope proteins (Com44/Cim44, Com70, and Cim97) in close physical proximity to a partially translocated chimeric precursor protein (Wu, C., Seibert, F. S., and Ko, K. (1994) J. Biol. Chem. 269, 32264-32271). In this study we report the characterization of a cDNA clone encoding a member of the Com44/Cim44 envelope proteins. The combined data from nucleotide sequencing, and RNA and protein blot analyses indicate the existence of multiple forms of the 44-kDa envelope protein. Depending on the plant species examined, immunologically-related protein bands with molecular masses of 42 to 46 kDa were observed. Organelle subfractionation, protease treatment, and immunomicroscopic studies together provide an indication that the immunologically-related proteins may be present in both the outer and inner envelope membranes. Co-migration of the product synthesized from the cDNA insert with a 44-kDa immunoreactive band of the chloroplast envelope, and the in vitro import results, together suggest that the in vitro synthesized 44-kDa protein is targeted to the envelope membrane without any further processing.

Isolation and characterization of a cDNA clone encoding a cognate 70-kDa heat shock protein of the chloroplast envelope.

The translocation of proteins into the endoplasmic reticulum, the mitochondrion, and the chloroplast has recently been shown to involve homologues of the highly conserved 70-kDa heat shock protein (HSP70) family. In this study, we have isolated and sequenced a full-length cDNA clone encoding a cognate 70-kDa heat shock protein of the spinach chloroplast envelope (SCE70). The cDNA insert is 2,535 base pairs long and codes for 653 amino acid residues of a protein with a predicted molecular mass of 71,731 daltons. The deduced amino acid sequence shows a high degree of homology with HSP70 proteins from other organisms. Southern genomic and RNA analyses reveal different hybridization patterns than that observed for a heat-inducible 70-kDa protein gene. The protein synthesized from the SCE70 cDNA insert co-migrates with a 70-kDa polypeptide of the chloroplast envelope following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot analysis and import studies indicate that SCE70 is associated with the chloroplast outer envelope. The import data suggest that SCE70 is targeted to the envelope membrane via a pathway different from other plastidic precursors but similar to that recently reported for outer envelope proteins SOE1 and OM14.