NTPs , and results in the irreversible Jennifer Davila-Aponte and interaction of precursors with the chloroplastic envelopes. Kenneth Keegstra 1At this stage, the precursor remains susceptible to exo-MSU-DOE Plant Research Laboratory, Michigan State University, genous protease and the transit peptide is not cleaved East Lansing, MI 48824, USA by the stromal processing peptidase, indicating that the 1 Corresponding author precursor has not completely traversed the envelope membranes (Cline et al., 1985). Translocation of precursors Cytoplasmically synthesized precursors interact with across the envelope membranes can be initiated by raising translocation components in both the outer and inner stromal ATP concentrations (Pain and Blobel, 1987; Theg envelope membranes during transport into chloro et al., 1989). After a precursor has traversed the envelope plasts. Using co-immunoprecipitation techniques, with membranes, the transit peptide is proteolytically removed antibodies specific to known translocation components, by a stromal processing peptidase, producing a maturewe identified stable interactions between precursor sized protein in the stromal compartment (Reed et al., proteins and their associated membrane translocation 1990). components in detergent-solubilized chloroplasticTranslocation of precursors across the two chloroplastic membrane fractions. Antibodies specific to the outer envelope membranes is thought to occur simultaneously envelope translocation components OEP75 and OEP34, at 'contact sites' (Schnell and Blobel, 1993), a term given the inner envelope translocation component IEP110 to regions where both envelope membranes are found in and the stromal Hsp100, ClpC, specifically co-immunoclose physical proximity. By analogy with mitochondria, precipitated precursor proteins under limiting ATP where precursors must also cross two membranes, preconditions, a stage we have called docking. A portion cursors at contact sites are thought to interact with proteinof these same translocation components was coaceous complexes from both the inner and outer immunoprecipitated as a complex, and could also be membranes (for review, see Schatz and Dobberstein, detected by co-sedimentation through a sucrose density 1996). In mitochondria, translocation complexes from the gradient. ClpC was observed only in complexes with outer and inner membranes can act independently from those precursors utilizing the general import apparone another, forming contact sites only when precursors atus, and its interaction with precursor-containing associate with both complexes simultaneously (Segui-Real translocation complexes was destabilized by ATP. et al., 1993;Horst et al., 1995). Whether simultaneous Finally, ClpC was co-immunoprecipitated with a porengagement is required in chloroplasts is presently tion of the translocation components of both outer and unknown. inner envelope membranes, even in the absence of Recent work on the chloroplastic protein import apparadded precursors. We discuss possible roles for stromal atus...

The function of Tic40 during chloroplast protein import was investigated. Tic40 is an inner envelope membrane protein with a large hydrophilic domain located in the stroma. Arabidopsis null mutants of the atTic40 gene were very pale green and grew slowly but were not seedling lethal. Isolated mutant chloroplasts imported precursor proteins at a lower rate than wild-type chloroplasts. Mutant chloroplasts were normal in allowing binding of precursor proteins. However, during subsequent translocation across the inner membrane, fewer precursors were translocated and more precursors were released from the mutant chloroplasts. Cross-linking experiments demonstrated that Tic40 was part of the translocon complex and functioned at the same stage of import as Tic110 and Hsp93, a member of the Hsp100 family of molecular chaperones. Tertiary structure prediction and immunological studies indicated that the C-terminal portion of Tic40 contains a TPR domain followed by a domain with sequence similarity to co-chaperones Sti1p/Hop and Hip. We propose that Tic40 functions as a cochaperone in the stromal chaperone complex that facilitates protein translocation across the inner membrane.

Transport of cytoplasmically synthesized proteins into chloroplasts uses an import machinery present in the envelope membranes. To identify the components of this machinery and to begin to examine how these components interact during transport, chemical cross-linking was performed on intact chloroplasts containing precursor proteins trapped at a particular stage of transport by ATP limitation. Large crosslinked complexes were observed using three different reversible homobifunctional cross-linkers. Three outer envelope membrane proteins (OEP86, OEP75, and OEP34) and one inner envelope membrane protein (IEP110), previously reported to be involved in protein import, were identified as components of these complexes. In addition to these membrane proteins, a stromal member of the hsp100 family, ClpC, was also present in the complexes. We propose that ClpC functions as a molecular chaperone, cooperating with other components to accomplish the transport of precursor proteins into chloroplasts. We also propose that each envelope membrane contains distinct translocation complexes and that a portion of these interact to form contact sites even in the absence of precursor proteins.

NAD is a ubiquitous coenzyme involved in oxidation-reduction reactions and is synthesized by way of quinolinate. Animals and some bacteria synthesize quinolinate from tryptophan, whereas other bacteria synthesize quinolinate from aspartate (Asp) using l-Asp oxidase and quinolinate synthase. We show here that Arabidopsis (Arabidopsis thaliana) uses the Asp-to-quinolinate pathway. The Arabidopsis l-Asp oxidase or quinolinate synthase gene complemented the Escherichia coli mutant defective in the corresponding gene, and T-DNA-based disruption of either of these genes, as well as of the gene coding for the enzyme quinolinate phosphoribosyltransferase, was embryo lethal. An analysis of functional green fluorescent protein-fused constructs and in vitro assays of uptake into isolated chloroplasts demonstrated that these three enzymes are located in the plastid.

Proteins from both the inner and outer envelope membranes are engaged in the recognition and translocation of precursor proteins into chloroplasts. A 110 kDa protein of the chloroplastic inner envelope membrane was identified as a component of the protein import apparatus by two methods. First, this protein was part of a 600 kDa complex generated by cross‐linking of precursors trapped in the translocation process. Second, solubilization with detergents of chloroplasts containing trapped precursors resulted in the identification of a complex containing both radiolabeled precursor and IEP110. Trypsin treatment of intact purified chloroplasts was used to study the topology of IEP110. The protease treatment left the inner membrane intact while simultaneously degrading domains of inner envelope proteins exposed to the intermembrane space. About 90 kDa of IEP110 was proteolitically removed, indicating that large portions protrude into the intermembrane space. Hydropathy analysis of the protein sequence deduced from the isolated cDNA clone in addition to Western blot analysis using an antiserum of IEP110 specific to the N‐terminal 20 kDa, suggests that the N‐terminus serves to anchor the protein in the membrane. We speculate that IEP110 could be involved in the formation of translocation contact sites due to its specific topology.

Three components of the chloroplast protein translocon, Tic110, Hsp93 (ClpC), and Tic40, have been shown to be important for protein translocation across the inner envelope membrane into the stroma. We show the molecular interactions among these three components that facilitate processing and translocation of precursor proteins. Transit-peptide binding by Tic110 recruits Tic40 binding to Tic110, which in turn causes the release of transit peptides from Tic110, freeing the transit peptides for processing. The Tic40 C-terminal domain, which is homologous to the C terminus of cochaperones Sti1p/Hop and Hip but with no known function, stimulates adenosine triphosphate hydrolysis by Hsp93. Hsp93 dissociates from Tic40 in the presence of adenosine diphosphate, suggesting that Tic40 functions as an adenosine triphosphatase activation protein for Hsp93. Our data suggest that chloroplasts have evolved the Tic40 cochaperone to increase the efficiency of precursor processing and translocation.

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Transport of cytoplasmically synthesized precursor proteins into chloroplasts, like the protein transport systems of mitochondria and the endoplasmic reticulum, appears to require the action of molecular chaperones. These molecules are likely to be the sites of the ATP hydrolysis required for precursor proteins to bind to and be translocated across the two membranes of the chloroplast envelope. Over the past decade, several different chaperones have been identified, based mainly on their association with precursor proteins and/or components of the chloroplast import complex, as putative factors mediating chloroplast protein import. These factors include cytoplasmic, chloroplast envelope-associated and stromal members of the Hsp70 family of chaperones, as well as stromal Hsp100 and Hsp60 chaperones and a cytoplasmic 14-3-3 protein. While many of the findings regarding the action of chaperones during chloroplast protein import parallel those seen for mitochondrial and endoplasmic reticulum protein transport, the chloroplast import system also has unique aspects, including its hypothesized use of an Hsp100 chaperone to drive translocation into the organelle interior. Many questions concerning the specific functions of chaperones during protein import into chloroplasts still remain that future studies, both biochemical and genetic, will need to address.