Chloroplasts are a major destination of protein traffic within leaf cells.

Chloroplasts are a major destination of protein traffic within leaf cells. into chloroplasts. Except for some outer envelope membrane proteins, nucleus-encoded chloroplast proteins are synthesized as higher molecular excess weight precursors with N-terminal extensions called transit peptides. Transit peptides are necessary and adequate for the import of precursor proteins into chloroplasts. Transport across the double membrane envelope is definitely mediated by a set of translocon parts located in the envelope. Several translocon parts have been recognized from pea chloroplasts by cross-linking or coimmunoprecipitating with importing precursor proteins (examined by Schleiff and Soll, 2000). They may be collectively named Toc (for translocon in the outer envelope membrane of chloroplasts) buy AZD6244 and Tic (for translocon in the inner envelope membrane of chloroplasts) proteins (Schnell et al., 1997). Among the Toc Mmp2 parts recognized, Toc159 is proposed to buy AZD6244 function as the transit peptide receptor (Perry and Keegstra, 1994; Ma et al., 1996). A considerable amount of evidence shows that Toc75 is the major component of the protein-translocating channel in the outer membrane (Hinnah et al., 1997; Reumann et al., 1999). The function of Toc34 is not clear. It has been shown to be tightly associated with Toc75 (Seedorf et al., 1995). Arabidopsis offers two Toc34 orthologs, atToc34 and atToc33. These two proteins seem to have unique but overlapping functions (Gutensohn et al., 2000). Techniques such as coimmunoprecipitation and cross-linking, when used to identify translocon parts, usually determine abundant and stably connected parts. Regulatory components that are present in minute amounts or only transiently, and upstream regulators present in different locations, usually are missed by these techniques. More recently, genetics has been used to study protein import into chloroplasts. Arabidopsis mutants have been found for two translocon components, atToc159 (Bauer et al., 2000) and atToc33 (Jarvis et al., 1998). These mutants confirmed that the translocon components identified by cell biology techniques function in chloroplast import. They also provided valuable information on the physiology and regulation of chloroplast protein import. Furthermore, maize and Arabidopsis mutants defective in protein transport to the thylakoid also have been isolated (Voelker and Barkan, 1995; Voelker et al., 1997; Roy and Barkan, 1998; Amin et al., 1999; Moore et al., 2000). These mutants helped to further define the various pathways of transport to the thylakoid and even helped to uncover a new export pathway in encodes a nuclear protein that positively regulates the transcript abundance of and in leaves. RESULTS Screening Strategy and Mutant Isolation For mutant isolation, we designed and transformed into wild-type Arabidopsis a reporter construct, TP30, to use as a selection marker. As shown in Figure 1A, TP30 encoded two fusion proteins. The first fusion protein was the transit peptide of the small subunit of ribulose bisphosphate carboxylase (RBCS) fused to hygromycin phosphotransferase (HPT). The second fusion protein contained the maize transit peptide fused to the -glucuronidase (GUS) (Kl?sgen et al., 1989). Both buy AZD6244 fusion constructs were placed under the control of the 35S promoter of mutations that affect the expression of the transgene. However, because of technical difficulties of observing GUS staining in chloroplasts, this marker was not used in the actual screens. Open in a separate window Figure 1. Screening of Mutants. (A) Scheme of the TP30 construct. LB and RB, left and right borders of the T-DNA, respectively; 35S, the 35S promoter of + TP30, a mutant buy AZD6244 line containing the TP30 marker. Plants were grown on medium containing 60 g/mL hygromycin for 26 days. (D) The visible phenotype of mutant is defective in chloroplast protein import. Chloroplasts were isolated from 28-day-old plants and useful for in vitro proteins transfer tests with precursor protein of RBCS and plastocyanin (Personal computer). Open up triangles reveal the precursor proteins, and shut circles reveal the imported adult proteins. WT, crazy type. To show the feasibility of our display, we crossed the TP30 create in to the known chloroplast proteins transfer mutant mutant including the TP30 reporter was certainly even more hygromycin resistant.