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Effects of CURT1 on Grana Stacking

Graduate student Eric Waddell

Graduate student Eric Waddell

Plants, cyanobacteria, and green algae rely on photosynthesis for energy and sugar production. Chloroplast, the organelle where photosynthesis occurs, is specifically organized in higher plantae to allow for efficient photosynthesis. This structure consists of an outer and inner envelope, stroma, thylakoid membrane, and lumen. The Thylakoid membrane is further organized into grana, round disk shaped membrane structures that stack 5-20 disks high. Inside the grana and thylakoid membrane of a chloroplast, is a continuous lumen. The grana and thylakoid membranes is connected by stroma lamellae. The grana and thylakoid membrane structure is important for separation of photosynthetic proteins. Photosystem I (PSI) and Cytochrome b6/f (Cyt b6/f) complex are located along the stacked grana membranes, while Photosystem II (PSII) and ATP synthase (ATPase) are localized to the stroma-facing thylakoid membranes. The structure of the the stacked grana and physical separation of the photosynthesis complexes allows for a higher degree of control in the photosynthetic process, for example separation of PSI and PSII allows for the plant to control transfer excess excitation energy.

CURT1b, as identified in this paper, has previously been described as TMP14, thylakoid membrane phosphoprotein 14kD, and was thought to be a subunit of PSI. Previous research also found homologous genes across many plant, cyanobacteria, and green algae species. This new class of genes was named PSI-P, photosystem I phosphoproteins. Armbruster et al discovered a family of genes in Arabidopsis thaliana using co-expression of the known genes involved in photosynthesis. The unknown genes pulled from the co-expression data where then grouped hierarchically to determine their association with known photosynthetic genes. CURT1a, b, and c all grouped with other photosynthetic genes involved in PSI. However, the fourth gene (CURT1d) did not cluster with photosynthesis related genes. The predicted structure of the CURT1 gene family was determined by the sequence of the 4 homologs. They consist of an N-terminal amphipathic helix, two transmembrane alpha-helices, and a c-terminal coiled coil helix. The four helix domains are relatively conserved across different species, with the exception of the amphipathic helix. Intriguingly, the 4 domains of the CURT1 gene family are also found to be incorporated into aminoacyl-tRNA synthetases, aaRSs, in certain cyanobacteria.

Localization of the CURT1 proteins, as examined by staining of cell cultures, showed localization of CURT1s to the chloroplasts. Isolations of thylakoid, inner and outer envelope, and stroma from chloroplasts were run on gel blots and antibodies for three control proteins with known localizations, and antibodies to curt1a b and c where shown; all three localize to the thylakoid membrane. Endogenous curt1d expression is too low for western blot assays from tissue isolates, so they looked at a HA tagged over expressing line for CURT1d and found that it to is located in the thylakoid membrane. Membrane association of the CURT1s was studied using chaotropic salts to disassociate membrane bound proteins. Curt1b and CURT1d behaved similarly to PetC, a control with 1 transmembrane domain, while CURT1a and c behaved similarly to Lhcb1 and Cyt b6, which have 3 and 4 transmembrane domains.

Using a fusion of maltose binding protein for extraction, the researchers studied the endogenous protein concentrations of CURT1s. They found CURT1a to be the most abundant, and CURT1d least abundant. From previously generated insertion mutants (knockouts), they generated double, triple, and quadruple knockouts of the CURT1 genes. Interestingly, the knockout of CURT1a had a negative effect on the presence of CURT1b and CURT1c. The double mutants had a superadditive effect on the remaining CURT1 protein. And the triple knockouts showed no presence of CURT1 protein. Seven CURT1 dimer complexes where discovered using native blue page assay and sds page to separate out the interacting subunits. Homo and hetero dimers/trimers of CURT1a and b were found using a western blot assay with antibodies for each.

Photosynthetic efficiency is reduced in knockout mutants, with quantum yield and non-photochemical quenching reduced in CURT1a knockouts and drastically reduced in double, triple, and quadruple knockouts. Initial fluorescence from the PSI is increased in the initial time after light exposure from the resting state and WT can better incorporate light energy 100-200ms after exposure.  Compared to WT, the knockout mutants are impaired in cyclic electron transport.

There is a striking physiological change in knockout and overexpressing mutants. In knockout mutants we see a reduction of grana stack formation, which increases in severity with the inclusion of multiple CURT1 knockouts. Alternatively, when we overexpress CURT1 we see increased numbers of grana stacks. In triple and quadruple knockouts we see the introduction of unique vacuole-like structures between the layers of the thylakoid membrane. SEM, of chloroplasts with the outer protective membrane stripped away, showed a top-down version of the physiological changes seen previously. It also, provided a better representation of the elongated thylakoid membrane in the quadruple CURT1 knockout.

SEM and gold immunoblotting (above) for CURT1a was used to detect the areas of the thylakoid membrane that contained CURT1 proteins. The results show that the margins, or periphery, of the grana stacks contained the highest proportion of CURT1 protein. This suggested that CURT1s were responsible for the induction of thylakoid membrane curvature. To verify that the CURT1 proteins could cause curvature in a membrane, in vitro studies were conducted. The results showed that there was tubule formation in artificial lipid membranes. It also showed oligomerization of the CURT1 proteins.

The researchers provided a hypothetical model for how CURT1 proteins lead to grana stack formation in vivo. They suggest that the process is regulated by phosphorylation and subsequent transcriptional control of tap38/pph1. Phosphorylation, increases grana stacking, whereas dephosphorylation reduces grana stack formation.

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Armbruster, U., Labs, M., Pribil, M., Viola, S., Xu, W., Scharfenberg, M., … Leister, D. (2013). Arabidopsis CURVATURE THYLAKOID1 Proteins Modify Thylakoid Architecture by Inducing Membrane Curvature. The Plant Cell, 25(7), 2661–2678. http://doi.org/10.1105/tpc.113.113118