Protein import-independent functions of Tic56, a component of the 1-MDa translocase at the inner chloroplast envelope membrane

ABSTRACT

Tic56 is an essential subunit of a 1-MDa protein complex at the inner chloroplast envelope membrane that comprises Tic100, Tic214 and the protein conducting channel protein Tic20-I. The complex was characterized as the “general protein import translocase” because mutants in either of its subunits have a severe growth phenotype and fail to assemble a photosynthetic machinery. In a recent publication we show that the albino phenotype of tic56–1 mutants results at least in part from a defect in ribosome assembly and a deficiency in plastid translation. We furthermore could not detect any impairment of protein import activity with plastids from tic56–3 mutants, despite a lack of full-length Tic56 and a decreased abundance of other 1-MDa complex subunits. These findings suggest that the 1-MDa complex consists of subunits that have functions other than protein import.

Chloroplasts must import a large number of nucleus-encoded proteins after their translation in the cytosol to sustain their biochemical activities and biologic functions. This transport is catalyzed by protein complexes at the outer (TOC) and the inner chloroplast envelope membrane (TIC).^1-3 In an effort to define the interaction network of the TOC translocon component Toc159, we isolated several components of the inner envelope membrane TIC complexes among them Tic56, Tic20, Tic214 and Tic100 using TAP-tagged Toc159 as a bait.⁴ These proteins constitute a 1-MDa complex at the inner chloroplast envelope membrane that forms a protein conducting channel for plastid protein import.⁵ Mutants in individual components of the 1-MDa complex fail to assembly the entire complex and show a severe growth phenotype and a deficiency in photosynthetic protein accumulation. Because Tic20-I is predominantly expressed in photosynthetic tissues, it was concluded that the 1-MDa complex represents the general import translocon at the inner envelope membrane of chloroplasts that is specifically required for the import of photosynthetic proteins.^5,6

In a series of 2 recent papers we characterized Tic56 and the 1-MDa complex further.^4,7 We made use of 2 mutant alleles; tic56–1 that completely lacks Tic56 and fails to accumulate the 1-MDa complex and tic56–3 that expresses low amounts of truncated and elongated forms of Tic56.^4,7 While the elongated form resides outside of plastids as indicated by its sensitivity against thermolysin⁴ the truncated form localizes inside plastids and is incorporated into the 1-MDa complex; albeit at decreased abundance compared with wildtype.^5,7 Even though both mutant lines are affected in the accumulation of Tic56, tic56–1 lines are albino and cannot grow autotrophically while tic56–3 plants do not show any significant defect in photosynthesis and have a pale-green phenotype.^4,5

The weak phenotype of tic56–3 plants is striking because we expected that a significant decrease in the abundance of essential 1-MDa complex subunits would result in constraints on the import of photosynthetic proteins under conditions of active photosynthesis. We therefore analyzed the import properties of tic56–3 plastids in controlled in vitro import experiments using the precursors of PDH, RbcS and SSR16. Although these precursors cover different functional categories of chloroplast biology, we were unable to observe any effect on protein import into tic56–3 plastids.^4,7 These results were verified in protoplast assays.⁴ Notably, tic56–3 and tic56–1 differ in the accumulation of Tic20-I, that accumulates to higher levels in the tic56–3 mutant compared with tic56–1.⁷ It is therefore likely that Tic20-I sustains plastid protein import in the tic56–3 mutant to an extent that makes it indistinguishable from wildtype in controlled in vitro import experiments.⁷ In this scenario, the Tic20-I containing 1-MDa complex is required at early stages of development, and individual subunits of this complex -as in the case of Tic56- are partially dispensable at later developmental stages.

Despite their severe growth phenotype, tic56-I mutants are capable of importing a large number of proteins into plastids suggesting that import occurs independent of the 1-MDa complex.⁴ A similar conclusion was recently reached from experiments in which translation of the plastid encoded Tic214 (YCF1) subunit of the 1-MDa complex was blocked with Spectinomycin.⁸ Thus there is clearly an alternative import pathway, i.e. the term “general import pathway” is misleading. The additional import pathway is potentially sustained by Tic20-IV, an isoform of Tic20-I that is the only TIC associated protein that is significantly upregulated in the tic56–1 mutant at the transcriptional level.⁷ It was suggested that the additional import pathway is responsible for the import of housekeeping proteins,⁶ but its client protein specificity in distinction to the client protein specificity of the “general import pathway” remains obscure because the latter was not addressed experimentally.

To fill this void, we performed correlative quantitative proteome analyses of tic56–1, ppi2 and wildtype plastids and compared the data with an albino plastid reference proteome that was acquired to distinguish common properties of unrelated albino plants at the proteome level from specific defects of a mutation.⁹ This comparison detected a significant defect in ribosome assembly that is specific for the 2 import mutants tic56–1 and ppi2, and accompanied by a diminished expression of plastid encoded proteins.⁷ From a developmental perspective, a plastid translation system is required before the assembly of a photosynthetic machinery, thus the albino phenotype of tic56–1 and ppi2 is most likely a result of a defect in plastid translation. This is consistent with a necessity of Tic56 at early developmental stages and supported by the fact that Spectinomycin-treatment of wildtype plants phenocopies the molecular phenotype of tic56–1 plants at the proteome level.⁷ Thus, a distinction of client protein preference between “housekeeping proteins” and “photosynthetic proteins” is not meaningful for ppi2 or tic56–1.

It is conceivable that the defect in ribosome assembly is caused by an import defect in tic56–1 plants, however, there is a striking co-expression of genes involved in the processing of rRNAs with Tic56.⁷ Even though Tic56 is thought to localize to the intermembrane space,⁵ a function in rRNA processing and ribosome assembly cannot be excluded at this point. Notably, recent data showed that Tic214 (YCF1) is involved in controlling the biogenesis of NDH, PSI and Cytb₆f complexes at the thylakoid membrane. YCF1 was found associated with these complexes supporting dual localization of YCF1 in thylakoid and inner envelope membranes.¹⁰ Thus the 1-MDa complex may have a modular structure with its constituents contributing to different functions within the chloroplast. Such a scenario is supported by the observation, that Tic56 accumulates to almost wildtype levels in tic20-I mutants.⁷

In conclusion, our recent publications provided new insights into functions of 1-MDa complex subunits and suggests a molecular basis for the albino phenotype of its mutants. We furthermore demonstrate that the application of large-scale proteomics tools to complex biologic questions opens up unprecedented possibilities to extract unbiased information from the correlative comparison of different “proteotypes."

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by the DFG under Grant number Ba1902/3–1.