Initial binding of preproteins involving the Toc159 receptor can be bypassed during protein import into chloroplasts

Two integral outer envelope GTPases, Toc34 and Toc86, are proposed to regul ate the recognition and translocation of nuclear-encoded preproteins during the early stages of protein import into chloroplasts. Defining the precise roles of Toc86 and Toc34 has been complicated by the inability to distingu ish their GTPase activities. Furthermore, the assignment of Toc86 function is rendered equivocal by recent reports suggesting that the standard protoc ol for the isolation of chloroplasts results in significant proteolysis of Toc86 (B. Bolter, T. May, J. Soll [1998] FEBS Lett 441: 59-62; G. Schatz [1 998] Nature 395: 439-440). We demonstrate that Toc86 corresponds to a nativ e protein of 159 kD in pea (Pisum sativum), designated Toc159. We take adva ntage of the proteolytic sensitivity of Toc159 to selectively remove its 10 0 kD cytoplasmic GTPase domain and thereby distinguish its activities from other import components. Proteolysis eliminates detectable binding of prepr oteins at the chloroplast surface, which is consistent with the proposed ro le of Toc159 as a receptor component. Remarkably, preprotein translocation across the outer membrane can occur in the absence of the Toc159 cytoplasmi c domain, suggesting that binding can be bypassed. Translocation remains se nsitive to GTP analogs in the absence of the Toc159 GTP-binding domain, pro viding evidence that Toc34 plays a key role in the regulation of translocat ion by GTP.