Chloroplasts are a major destination of protein traffic within leaf cells. Protein import into chloroplasts is mediated by a set of translocon complexes at the chloroplast envelope. Current data indicate that the expression of translocon genes is regulated in a tissue-specific manner, possibly to accommodate the higher import demand of chloroplasts in leaves and the lower demand of plastids in other tissues. We have designed a transgene-based positive screen to isolate mutants disrupted in protein import into plastids. The first locus we isolated, CIA2 , encodes a protein containing a motif conserved within the CCT family of transcription factors. Biochemical analysis indicates that CIA2 is responsible for specific upregulation of the translocon genes atToc33 and atToc75 in leaves. Identification of CIA2 provides new insights into the tissue-specific regulation of translocon gene expression. INTRODUCTIONMost proteins in chloroplasts are encoded by the nucleus and imported post-translationally into chloroplasts. Except for some outer envelope membrane proteins, nucleusencoded chloroplast proteins are synthesized as higher molecular weight precursors with N-terminal extensions called transit peptides. Transit peptides are necessary and sufficient for the import of precursor proteins into chloroplasts. Transport across the double membrane envelope is mediated by a set of translocon components located in the envelope. Several translocon components have been identified from pea chloroplasts by cross-linking or coimmunoprecipitating with importing precursor proteins (reviewed by Schleiff and Soll, 2000). They are collectively named Toc (for translocon at the outer envelope membrane of chloroplasts) and Tic (for translocon at the inner envelope membrane of chloroplasts) proteins (Schnell et al., 1997).Among the Toc components identified, Toc159 is proposed to function as the transit peptide receptor (Perry and Keegstra, 1994;Ma et al., 1996). A considerable amount of evidence indicates that Toc75 is the major component of the protein-translocating channel in the outer membrane (Hinnah et al., 1997;Reumann et al., 1999). The function of Toc34 is not clear. It has been shown to be tightly associated with Toc75 (Seedorf et al., 1995). Arabidopsis has two Toc34 orthologs, atToc34 and atToc33. These two proteins seem to have distinct but overlapping functions (Gutensohn et al., 2000).Techniques such as coimmunoprecipitation and crosslinking, when used to identify translocon components, usually identify abundant and stably associated components. Regulatory components that are present in minute amounts or only transiently, and upstream regulators present in different locations, usually are missed by these techniques.More recently, genetics has been used to study protein import into chloroplasts. Arabidopsis mutants have been found for two translocon components, atToc159 (Bauer et al., 2000) and atToc33 (Jarvis et al., 1998). These mutants confirmed that the translocon components identified by cell biology techniques functi...

Chloroplasts are a major destination of protein traffic within leaf cells. Protein import into chloroplasts is mediated by a set of translocon complexes at the chloroplast envelope. Current data indicate that the expression of translocon genes is regulated in a tissue-specific manner, possibly to accommodate the higher import demand of chloroplasts in leaves and the lower demand of plastids in other tissues. We have designed a transgene-based positive screen to isolate mutants disrupted in protein import into plastids. The first locus we isolated, CIA2 , encodes a protein containing a motif conserved within the CCT family of transcription factors. Biochemical analysis indicates that CIA2 is responsible for specific upregulation of the translocon genes atToc33 and atToc75 in leaves. Identification of CIA2 provides new insights into the tissue-specific regulation of translocon gene expression.

Sequence analysis of a newly identified polyubiquitin gene (UBQ13) from the Columbia ecotype of Arabidopsis thaliana revealed that the gene contained a 3.9-kb insertion in the coding region. All subclones of the 3.9-kb insert hybridized to isolated mitochondrial DNA. The insert was found to consist of at least two, possibly three, distinct DNA segments from the mitochondrial genome. A 590-bp region of the insert is nearly identical to the Arabidopsis mitochondrial nad1 gene. UBQ13 restriction fragments in total cellular DNA from ecotypes Ler, No-0, Be-0, WS, and RLD were identified and, with the exception of Be-0, their sizes were equivalent to that predicted from the corresponding ecotype Columbia UBQ13 restriction fragment without the mitochondrial insert. Isolation by polymerase chain reaction and sequence determination of UBQ13 sequences from the other ecotypes showed that all lacked the mitochondrial insert. All ecotypes examined, except Columbia, contain intact open reading frames in the region of the insert, including four ubiquitin codons which Columbia lacks. This indicates that the mitochondrial DNA in UBQ13 in ecotype Columbia is the result of an integration event that occurred after speciation of Arabidopsis rather than a deletion event that occurred in all ecotypes except Columbia. This stable movement of mitochondrial DNA to the nucleus is so recent that there are few nucleotide changes subsequent to the transfer event. This allows for precise analysis of the sequences involved and elucidation of the possible mechanism. The presence of intron sequences in the transferred nucleic acid indicates that DNA was the transfer intermediate. The lack of sequence identity between the integrating sequence and the target site, represented by the other Arabidopsis ecotypes, suggests that integration occurred via nonhomologus recombination. This nuclear/organellar gene transfer event is strikingly similar to the experimentally accessible process of nuclear integration of introduced heterologous DNA.

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The Arabidopsis thaliana ecotype Columbia ubiquitin gene family consists of 14 members that can be divided into three types of ubiquitin genes; polyubiquitin genes, ubiquitin-like genes and ubiquitin extension genes. The isolation and characterization of eight ubiquitin sequences, consisting of four polyubiquitin genes and four ubiquitin-like genes, are described here, and their relationships to each other and to previously identified Arabidopsis ubiquitin genes were analyzed. The polyubiquitin genes, UBQ3, UBQ10, UBQ11 and UBQ14, contain tandem repeats of the 228-bp ubiquitin coding region. Together with a previously described polyubiquitin gene, UBQ4, they differ in synonymous substitutions, number of ubiquitin coding regions, number and nature of nonubiquitin C-terminal amino acid(s) and chromosomal location, dividing into two subtypes; the UBQ3/UBQ4 and UBQ10/UBQ11/UBQ14 subtypes. Ubiquitin-like genes, UBQ7, UBQ8, UBQ9 and UBQ12, also contain tandem repeats of the ubiquitin coding region, but at least one repeat per gene encodes a protein with amino acid substitutions. Nucleotide comparisons, Ks value determinations and neighbor-joining analyses were employed to determine intra- and intergenic relationships. In general, the rate of synonymous substitution is too high to discern related repeats. Specific exceptions provide insight into gene relationships. The observed nucleotide relationships are consistent with previously described models involving gene duplications followed by both unequal crossing-over and gene conversion events.