Chloroplasts are the organelles that define plants. Along with many other functions, these organelles are responsible for photosynthesis (the process whereby sunlight energy is harnessed to power the cellular activities of life), and consequently they are essential for plant growth, and for the myriad ecosystems that depend on plants.
The development of chloroplasts depends on the import of thousands of nucleus-encoded proteins from the cytosol [1,2]. Such import is mediated by multiprotein complexes in the chloroplast envelope membranes termed TOC and TIC (Translocon at the Outer/Inner membrane of Chloroplasts). The TIC complex mediates inner membrane transport, and provides the ATP-dependent driving force for the process. A critical component of the TIC complex is the co-chaperone Tic40, which regulates internal chaperones at the conclusion of the import process [3,4]. Upon exiting the TIC machinery, many imported proteins (e.g., components of the photosynthetic machinery) engage additional sorting pathways that direct them to the internal thylakoid membranes, where the light-dependent reactions of photosynthesis take place .
Mutant plants lacking the Tic40 protein are pale-yellow due to defective protein transport and retarded chloroplast development . To identify novel factors involved in chloroplast protein transport, we employed forward genetics: we screened for extragenic suppressors of the tic40 mutant. The suppressor mutants were considerably greener and more developed than tic40 plants, and they identified the STIC1 and STIC2 (SUPPRESSOR OF TIC40) genes. Recent work revealed that the STIC1 gene encodes a member of the Alb3/Oxa1/YidC family of membrane protein integrases, located in the thylakoid membrane, and that STIC2 encodes a protein of unknown function related to bacterial protein YbaB . Moreover, it was shown that the STIC1 and STIC2 proteins together define a novel protein-transport pathway leading from the TIC apparatus, at the envelope, to the thylakoid membranes. While this was a significant breakthrough, significant questions remain unanswered. For example, what are the clients of this STIC-dependent pathway? – in other words, which proteins does it deliver to the thylakoids? And, what other factors act in the STIC pathway? The latter is an important question as it seems highly unlikely that STIC1/2 act alone.
To address these questions, we propose to take two different proteomics approaches. The first question will be addressed using an isobaric tag-based quantitative proteomics approach called TMT (tandem mass tagging; Thermo Fisher) . In this work, chloroplasts will be isolated from wild-type and stic1 and stic2 mutant plants, and then an analysed using TMT to determine which proteins, specifically, are depleted in the mutants. Proteins found to be depleted in both stic mutants, relative to wild type, will be candidate clients of the STIC pathway, and will be verified as such in subsequent experimental analyses.
The second question will be addressed by applying an affinity purification approach called tandem affinity purification (TAP), or similar [7,8]. Chloroplasts isolated from plant lines expressing TAP-tagged STIC1 or STIC2 proteins (these plant lines have already been generated) will be subjected to gentle lysis and membrane-solubilization prior to affinity-purification of the STIC-containing complexes. The components of these purified STIC complexes will then be identified by mass spectrometry, paving the way for a range of molecular, genetic and cell biological approaches to elucidate the functions of the identified components, and of the STIC pathway generally.
At the conclusion of this project, it is expected that we will have a greatly improved level of understanding of an important new pathway of thylakoid protein biogenesis in plant chloroplasts.
1. Jarvis, P. and López-Juez, E. (2013) Biogenesis and homeostasis of chloroplasts and other plastids. Nat. Rev. Mol. Cell Biol. 14:787-802.
2. Jarvis, P. (2008) Targeting of nucleus-encoded proteins to chloroplasts in plants (Tansley Review). New Phytol. 179:257-285.
3. Kovacheva, S., Bédard, J., Patel, R., Dudley, P., Twell, D., Ríos, G., Koncz, C. and Jarvis, P. (2005) In vivo studies on the roles of Tic110, Tic40 and Hsp93 during chloroplast protein import. Plant J. 41:412-428.
4. Bédard, J., Kubis, S., Bimanadham, S. and Jarvis, P. (2007) Functional similarity between the chloroplast translocon component, Tic40, and the human co-chaperone, Hip. J. Biol. Chem. 282:21404-21414.
5. Bédard, J., Trösch, R., Wu, F., Ling, Q., Flores-Pérez, Ú., Töpel, M., Nawaz, F. and Jarvis, P. (2017) Suppressors of the chloroplast protein import mutant tic40 reveal a genetic link between protein import and thylakoid biogenesis. Plant Cell doi: 10.1105/tpc.16.00962. [Epub ahead of print].
6. Rose, C.M. et al. (2016) Highly multiplexed quantitative mass spectrometry analysis of ubiquitylomes. Cell Syst. 3:395-403.e4.
7. Rohila, J.S., Chen, M., Cerny, R. and Fromm, M.E. (2004) Improved tandem affinity purification tag and methods for isolation of protein heterocomplexes from plants. Plant J. 38:172-181.
8. Low, T.Y., Peng, M., Magliozzi, R., Mohammed, S., Guardavaccaro, D. and Heck, A.J. (2014) A systems-wide screen identifies substrates of the SCFβTrCP ubiquitin ligase. Sci Signal. 7(356):rs8.
Bédard, J., Trösch, R., Wu, F., Ling, Q., Flores-Pérez, Ú., Töpel, M., Nawaz, F. and Jarvis, P. (2017) Suppressors of the chloroplast protein import mutant tic40 reveal a genetic link between protein import and thylakoid biogenesis. Plant Cell doi: 10.1105/tpc.16.00962. [Epub ahead of print].
Flores-Pérez, Ú., Bédard, J., Tanabe, N., Lymperopoulos, P., Clarke, A.K. and Jarvis, P. (2016) Functional analysis of the Hsp93/ClpC chaperone at the chloroplast envelope. Plant Physiol. 170: 147-162.
Ling, Q. and Jarvis, P. (2015) Regulation of chloroplast protein import by the ubiquitin E3 ligase SP1 is important for stress tolerance in plants. Curr. Biol. 25:2527-2534.
Trösch, R., Töpel, M., Flores-Pérez, Ú. and Jarvis, P. (2015) Genetic and physical interaction studies reveal functional similarities between ALBINO3 and ALBINO4 in Arabidopsis. Plant Physiol. 169: 1292-1306.