Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the ...

Martin-Luther-Universitat Halle-Wittenberg

Two families of GTPases, the Toc34 and Toc159 GTPase families, take on the task of preprotein recognition at the translocon at the outer membrane of chloroplasts (TOC translocon). The major Toc159 family members have highly acidic N-terminal domains (A-domains) that are non-essential and so far have escaped functional characterization. But recently, interest in the role of the A-domain has strongly increased. The new data of three independent studies provide evidence that the Toc159 A-domain I) participates in preprotein selectivity, II) has typical features of intrinsically unfolded proteins and III) is highly phosphorylated and possibly released from the rest of the protein by a proteolytic event. This hints to a complex regulation of A-domain function that is important for the maintenance of the preprotein selectivity at the TOC translocons.

Modifications at the A-domain of the chloroplast import receptor Toc159.

Research interest in proteomics is increasingly shifting toward the reverse genetic characterization of gene function at the proteome level. In plants, several distinct gene defects perturb photosynthetic capacity, resulting in the loss of chlorophyll and an albino or pale-green phenotype. Because photosynthesis is interconnected with the entire plant metabolism and its regulation, all albino plants share common characteristics that are determined by the switch from autotrophic to heterotrophic growth. Reverse genetic characterizations of such plants often cannot distinguish between specific consequences of a gene defect from generic effects in response to perturbations in photosynthetic capacity. Here, we set out to define common and specific features of protein accumulation in three different albino/pale-green plant lines. Using quantitative proteomics, we report a common molecular phenotype that connects the loss of photosynthetic capacity with other chloroplast and cellular functions, such as protein folding and stability, plastid protein import, and the expression of stress-related genes. Surprisingly, we do not find significant differences in the expression of key transcriptional regulators, suggesting that substantial regulation occurs at the posttranscriptional level. We examine the influence of different normalization schemes on the quantitative proteomics data and report all identified proteins along with their fold changes and P values in albino plants in comparison with the wild type. Our analysis provides initial guidance for the distinction between general and specific adaptations of the proteome in photosynthesis-impaired plants.

Common and specific protein accumulation patterns in different albino/pale-green mutants reveals regulon organization at the proteome level.

Arabidopsis is the model plant of choice for large-scale proteome analyses, because its genome is well annotated, essentially free of sequencing errors, and relatively small with little redundancy. Furthermore, most Arabidopsis organs are susceptible to standard protein solubilization protocols making protein extraction relatively simple. Many different facets of functional plant proteomics were established with Arabidopsis such as mapping the subcellular proteomes of organelles, proteo-genomic peptide mapping, and numerous studies on the dynamic changes in protein modification and protein abundances. As most standard proteomics technologies are now routinely applied, research interest is increasingly shifting towards the reverse genetic characterization of gene function at the proteome level, i.e., by profiling the quantitative proteome of wild type in comparison with mutant plant tissue. We report here a simple, standardizable protocol for the large-scale comparative quantitative proteome characterization of different Arabidopsis organs based on normalized spectral counting and suggest a statistical framework for data interpretation. Based on existing organellar proteome maps, proteins can be assigned to organelles, thus allowing the identification of organelle-specific responses.

Arabidopsis proteomics: a simple and standardizable workflow for quantitative proteome characterization.

We report on the characterization of Tic56, a unique component of the recently identified 1-MD translocon at the inner envelope membrane of chloroplasts (TIC) in Arabidopsis (Arabidopsis thaliana) comprising Tic20, Tic100, and Tic214. We isolated Tic56 by copurification with Tandem Affinity Purification-tagged Toc159 in the absence of precursor protein, indicating spontaneous and translocation-independent formation of the translocon at the outer envelope membrane of chloroplasts (TOC) and TIC supercomplexes. Tic56 mutant plants have an albino phenotype and are unable to grow without an external carbon source. Using specific enrichment of protein amino termini, we analyzed the tic56-1 and plastid protein import2 (toc159) mutants to assess the in vivo import capacity of plastids in mutants of an outer and inner envelope component of the anticipated TOC-TIC supercomplex. Inboth mutants, we observed processing of several import substrates belonging to various pathways. Our results suggest that despite the severe developmental defects, protein import into Tic56-deficient plastids is functional to a considerable degree, indicating the existence of alternative translocases at the inner envelope membrane.

Characterization of chloroplast protein import without Tic56, a component of the 1-megadalton translocon at the inner envelope membrane of chloroplasts.

The biogenesis and maintenance of cell organelles such as mitochondria and chloroplasts require the import of many proteins from the cytosol, a process that is controlled by phosphorylation. In the case of chloroplasts, the import of hundreds of different proteins depends on translocons at the outer and inner chloroplast membrane (TOC and TIC, respectively) complexes. The essential protein TOC159 functions thereby as an import receptor. It has an N-terminal acidic (A-) domain that extends into the cytosol, controls receptor specificity, and is highly phosphorylated  However, kinases that phosphorylate the TOC159 A-domain to enable protein import have remained elusive. Here, using co-purification with TOC159 from , we discovered a novel component of the chloroplast import machinery, the regulatory kinase at the outer chloroplast membrane 1 (KOC1). We found that KOC1 is an integral membrane protein facing the cytosol and stably associates with TOC. Moreover, KOC1 phosphorylated the A-domain of TOC159 , and in mutant  chloroplasts, preprotein import efficiency was diminished.  seedlings had reduced survival rates after transfer from the dark to the light in which protein import into plastids is required to rapidly complete chloroplast biogenesis. In summary, our data indicate that KOC1 is a functional component of the TOC machinery that phosphorylates import receptors, supports preprotein import, and contributes to efficient chloroplast biogenesis.

Birgit Agne

Institut fur Biochemie und Biotechnologie

Martin-Luther-Universitat Halle-Wittenberg

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Birgit Agne

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Martin-Luther-Universitat Halle-Wittenberg

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Institut fur Biochemie und Biotechnologie