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Plant Physiol, March 2000, Vol. 122, pp. 813-822
Initial Binding of Preproteins Involving the Toc159 Receptor Can
Be Bypassed during Protein Import into Chloroplasts1
Kunhua
Chen,
Xuejun
Chen, and
Danny J.
Schnell*
Department of Biological Sciences, Rutgers, The State University of
New Jersey, 101 Warren Street, Newark, New Jersey 07102
Two integral outer envelope GTPases,
Toc34 and Toc86, are proposed to regulate 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 distinguish their
GTPase activities. Furthermore, the assignment of Toc86 function is
rendered equivocal by recent reports suggesting that the standard
protocol 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 [1998] Nature 395: 439-440). We demonstrate
that Toc86 corresponds to a native protein of 159 kD in pea
(Pisum sativum), designated Toc159. We take advantage of
the proteolytic sensitivity of Toc159 to selectively remove its 100-kD
cytoplasmic GTPase domain and thereby distinguish its activities from
other import components. Proteolysis eliminates detectable binding of
preproteins at the chloroplast surface, which is consistent with the
proposed role of Toc159 as a receptor component. Remarkably, preprotein
translocation across the outer membrane can occur in the absence of the
Toc159 cytoplasmic domain, suggesting that binding can be bypassed.
Translocation remains sensitive to GTP analogs in the absence of the
Toc159 GTP-binding domain, providing evidence that Toc34 plays a key role in the regulation of translocation by GTP.
1
This work was supported by grants from the
National Science Foundation (MCB-9722914 and BIR-94131980) and a
Charles and Johanna Busch Memorial Fund Award.
*
Corresponding author; e-mail schnell{at}andromeda.rutgers.edu;
fax 973-353-1082.
© 2000 American Society of Plant Physiologists
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