Elaborate mechanisms have evolved for the translocation of nucleus-encoded proteins across the plastid envelope membrane. Although putative components of the import apparatus have been identified biochemically, their role in import remains to be proven in vivo. An Arabidopsis mutant lacking a new component of the import machinery [translocon at the outer envelope membrane of chloroplasts (Toc33), a 33-kilodalton protein] has been isolated. The functional similarity of Toc33 to another translocon component (Toc34) implies that multiple different translocon complexes are present in plastids. Processes that are mediated by Toc33 operate during the early stages of plastid and leaf development. The data demonstrate the in vivo role of a translocon component in plastid protein import.

Most chloroplast outer-membrane proteins are synthesized at their mature size without cleavable targeting signals. Their insertion into the outer membrane is insensitive to thermolysin pretreatment of chloroplasts and does not require ATP. It has therefore been assumed that insertion of outer-membrane proteins proceeds through a different pathway from import into the interior of chloroplasts, which requires a thermolysin-sensitive translocon complex and ATP. Here, we show that a model outer-membrane protein, OEP14, competed with the import of a chloroplast interior protein, indicating that the two import pathways partially overlapped. Cross-linking studies showed that, during insertion, OEP14 was associated with Toc75, a thermolysin-resistant component of the outer-membrane protein-conducting channel that mediates the import of interior-targeted precursor proteins. Whereas almost no OEP14 inserted into protein-free liposomes, OEP14 inserted into proteoliposomes containing reconstituted Toc75 with a high efficiency. Taken together, our data indicate that Toc75 mediates OEP14 insertion, and therefore plays a dual role in the targeting of proteins to the outer envelope membrane and interior of chloroplasts.

Using a transgene-based screening, we previously isolated several Arabidopsis mutants defective in protein import into chloroplasts. Positional cloning of one of the loci, CIA1, revealed that CIA1 encodes Gln phosphoribosyl pyrophosphate amidotransferase 2 (ATase2), one of the three ATase isozymes responsible for the first committed step of de novo purine biosynthesis. The cia1 mutant had normal green cotyledons but small and albino/pale-green mosaic leaves. Adding AMP, but not cytokinin or NADH, to plant liquid cultures partially complemented the mutant phenotypes. Both ATase1 and ATase2 were localized to chloroplasts. Overexpression of ATase1 fully complemented the ATase2-deficient phenotypes. A T-DNA insertion knockout mutant of the ATase1 gene was also obtained. The mutant was indistinguishable from the wild type. A double mutant of cia1/ATase1-knockout had the same phenotype as cia1, suggesting at least partial gene redundancy between ATase1 and ATase2. Characterizations of the cia1 mutant revealed that mutant leaves had slightly smaller cell size but only half the cell number of wild-type leaves. This phenotype confirms the role of de novo purine biosynthesis in cell division. Chloroplasts isolated from the cia1 mutant imported proteins at an efficiency less than 50% that of wild-type chloroplasts. Adding ATP and GTP to isolated mutant chloroplasts could not restore the import efficiency. We conclude that de novo purine biosynthesis is not only important for cell division, but also for chloroplast biogenesis.De novo biosynthesis of the purine ring is essential for plant growth and development. The major products, AMP and GMP, are the building blocks for DNA and RNA. AMP, when converted into ATP, is the major energy source for multiple cellular processes. Several important coenzymes, e.g. NAD and FAD, are also derived from the same pathway. In nodules of N-fixing tropical legumes, such as soybean (Glycine max) and cowpea (Vigna unguiculata), the pathway also plays a dominant role in primary nitrogen metabolism. The activity of enzymes in the purine biosynthesis pathway is enhanced considerably in nodules compared to other tissues. Therefore, most studies of purine biosynthesis in plants have used these legume nodules as materials and focused on the function of purine biosynthesis in nitrogen assimilation (for review, see Smith and Atkins, 2002). Studies on the role of purine biosynthesis in normal plant physiology or in non-N-fixing plants have been relatively few.The location of purine biosynthesis within plant cells is still in dispute. The plant enzymes in the pathway are similar to those in Escherichia coli, except that each plant enzyme has an N-terminal extension that is presumed to function as an organelle-targeting signal (Smith and Atkins, 2002). Fractionation studies of nodules indicate that the pathway is located within plastids (Boland and Schubert, 1983;Shelp et al., 1983). However, recent reports indicate that the pathway is present in both mitochondria and plastids (Atkins et al., 1997) or in...