Analysis of the Interactions of Preproteins with the Import Machinery over the Course of Protein Import into Chloroplasts

doi:10.1083/jcb.139.7.1677

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© The Rockefeller University Press, 0021-9525/1997//1677 $5.00
The Journal of Cell Biology, Volume 139, Number 7, , 1997 1677-1685

Article

Analysis of the Interactions of Preproteins with the Import Machinery over the Course of Protein Import into Chloroplasts

Andrei Kouranov and Danny J. Schnell

Department of Biological Sciences, Rutgers University, Newark, New Jersey 07102

We have investigated the interactions of two nuclear-encodedpreproteins with the chloroplast protein import machinery atthree stages in import using a label-transfer crosslinkingapproach. During energy-independent binding at the outer envelopemembrane, preproteins interact with three known componentsof the outer membrane translocon complex, Toc34, Toc75, andToc86. Although Toc75 and Toc86 are known to associate withpreproteins during import, a role for Toc34 in preprotein bindingpreviously had not been observed. The interaction of Toc34with preproteins is regulated by the binding, but not hydrolysisof GTP. These data provide the first evidence for a direct role for Toc34 in import, and provide insights into the functionof GTP as a regulator of preprotein recognition. Toc75 and Toc86are the major targets of cross-linking upon insertion of preproteinsacross the outer envelope membrane, supporting the proposalthat both proteins function in translocation at the outer membraneas well as preprotein recognition. The inner membrane proteins,Tic(21) and Tic22, and a previously unidentified protein of14 kD are the major targets of crosslinking during the latestages in import. These data provide additional support forthe roles of these components during protein translocationacross the inner membrane. Our results suggest a defined sequenceof molecular interactions that result in the transport of nuclear-encodedpreproteins from the cytoplasm into the stroma of chloroplasts.

Abbreviations used in this paper: APDP, N-[4-(p-azidosalicylamido)butyl]-3'(2-pyridyldithio)propionamide; Tic, translocon at the inner envelope membrane of chloroplasts; Toc, translocon at the outer envelope membrane of chloroplasts.

Address all correspondence to Danny J. Schnell, Department ofBiological Sciences, Rutgers University, 101 Warren Street,Newark, NJ 07102. Tel.: (973) 353-1082. Fax: (973) 353-1007.E-mail: schnell{at}andromeda.rutgers.edu

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