Department of Biology and Environmental Sciences; University of Gothenburg; Gothenburg, Sweden

Department of Biology and Environmental Sciences; University of Gothenburg; Gothenburg, Sweden

We recently showed that a Rab protein, CPRabA5e (CP = chloroplast localized), is located in chloroplasts of Arabidopsis thaliana where it is involved in various processes, such as thylakoid biogenesis and vesicle transport. Using a yeast two-hybrid method, CPRabA5e was shown to interact with a number of chloroplast proteins, including the CURVATURE THYLAKOID 1A (CURT1A) protein and the light-harvesting chlorophyll a/b binding protein (LHCB1.5). CURT1A has recently been shown to modify thylakoid architecture by inducing membrane curvature in grana, whereas LHCB1.5 is a protein of PSII (Photosystem II) facilitating light capture. LHCB1.5 is imported to chloroplasts and transported to thylakoid membranes using the post-translational Signal Recognition Particle (SRP) pathway. With this information as starting point, we here discuss their subsequent protein-protein interactions, given by the literature and Interactome 3D. CURT1A itself and several of the proteins interacting with CURT1A and LHCB1.5 have relations to vesicle transport and thylakoid morphology, which are also characteristics of cprabA5e mutants. This highlights the previous hypothesis of an alternative thylakoid targeting pathway for LHC proteins using vesicles, in addition to the SRP pathway.

In the cytosol, Rab proteins have multiple functions and membrane transport by vesicles can be seen as one of their primary functions.1 The Rab protein CPRabA5e, a homolog to the Ypt31/32 proteins in Saccharomyces cerevisiae (yeast), is found in chloroplasts of Arabidopsis thaliana. In yeast, Ypt31/21 function in exo- and endocytosis through vesicle transport regulation and CPRabA5e complemented yeast mutants defective of Ypt31/32, suggesting a role of CPRabA5e in vesicle transport of chloroplasts.2 Knockout mutants (cprabA5e) showed accumulation of chloroplast vesicles at 4ᵒC and grana stacks were comparably lower than in wild-type Arabidopsis.2 Using yeast two-hybrid, CPRabA5e was observed to interact with 13 different chloroplast proteins, two of them being LHCB1.5 and the Putative subunit of photosystem I complex (PsaP)/Uncharacterized protein At4g01150,2 now characterized as CURT1A. The CURT1 protein family in Arabidopsis consists of chloroplast localized proteins (grouped into A, B, C and D) having four helices, two transmembrane regions, a hydrophilic and a hydrophobic side, respectively.3

Inside the chloroplast, an aqueous stroma surrounds thylakoid membranes, constituting grana (concentrating PSII) and stroma lamellae (with PSI and ATP synthase).4,5 In general, grana are about 300–600 nm in diameter and have up to 20 layers of thylakoid membranes.6 The stacking of grana is dependent on numerous processes, including lipid composition, but less is known how curvature of grana margins develops and is maintained. However, it was recently shown that CURT1 proteins form oligomers and affect the grana architecture. Loss of function mutants of CURT1 results in broad, flat lobe-like thylakoids whereas an overexpression of CURT1A results in higher grana stacks but at the expense of the diameter being more narrow.3 Interestingly, the double mutant curt1ac accumulated vesicles in chloroplasts, which were located both close to the envelope and within the stroma. Thus, it was concluded that CURT1 proteins are directly involved in grana membrane curvature.3

Interactome 3D, a web tool providing structural annotations of protein-protein interaction networks (http://interactome3d.irbbarcelona.org/), shows CURT1A and LHCB1.5 to interact with four and three proteins, respectively. CURT1A interacts with Reticulon-like protein B4 and B8 (RTNLB4 and RTNLB8), Putative uncharacterized protein/At3g29270 and Regulator of G-protein signaling 1 (RGS1) protein, and LHCB1.5 interacts with Transcription factor TCP14 (TCP14), protein SNOWY COTYLEDON 2 (SCO2) and RGS1 (Fig. 1, Table 1). Notably, both CURT1A and LHCB1.5 interact with RSG1. RGS1 is found in the plasma membrane of Arabidopsis where it serves as a GTPase activating protein (GAP) to the α subunit of heterotrimeric G-protein Guanine nucleotide-binding protein α-1 subunit (GPA1).7-9 Interestingly, GPA1 has also been found to interact with the THYLAKOID FORMATION 1 (THF1) protein,10 a protein required for normal vesicle formation during thylakoid biogenesis. Mutants of THF1 show a variegated leaf phenotype, where the plastids in non-green sectors show accumulation of vesicle and lack normal thylakoid arrangement.11

Protein interactors to CURT1A and LHCB1.5, as given by Interactome 3D. ARAMEMNON was used to predict subcellular location, and numbers indicate consensus scores from multiple prediction tools where a number above 10 was considered reliable

CP, chloroplast; MT, mitochondria; SEC, secretory pathway.

CPRabA5e is shown as interacting with LHCB1.5 and CURT1A using yeast two-hybrid (thick lines)2, and being merged together with yeast two-hybrid results for LHCB1.5 and CURT1A from Interactome 3D (thin lines).

Two of the proteins interacting with CURT1A, RTNLB4 and RTNLB8, belong to a group of proteins (reticulons) mostly characterized in mammals and yeast where they are implicated in a number of processes, including intracellular transport. Reticulons in plants (RTNLBs) are less known, but Arabidopsis has 21 AtRTNLB genes identified and protein homology infers similar roles in plants as in animals.12,13 AtRTNLB4 has been found in the endoplasmic reticulum (ER), adjacent to chloroplasts where ER formed a net like structure surrounding the chloroplast, and results suggest it to be involved in ER-chloroplast traffic.12,13 It has also been shown to interact with another Rab protein in Arabidopsis, AtRABE1a.14 Three RTNLBs have been predicted to be chloroplast localized: AtRTNLB17, AtRTNLB18, and AtRTNLB21,15 but notably none of these corresponds to the two identified by Interactome 3D.

According to the literature RGS1, RTNLB4 and RTNLB8 lack chloroplast location, however data achieved from Interactome 3D show interaction with the chloroplast localized protein CURT1A (Fig. 1). One explanation could be that yeast two-hybrid often is performed using cDNA libraries covering the total proteome. However, some proteins in plant cells evidently have dual locations but are considered exceptions.16 Therefore spatial segregation could be an indication of false positive results, or is reflecting the possibility that similar proteins (like the identified ones) are present within the given organelle. Regardless, it is interesting to note that several of the proteins identified, using yeast two-hybrid with CPRabA5e as bait2 or Interactome 3D, have relations to vesicles, thylakoid formation and/or GTP cycling.

In contrast to RGS1 and the reticulons, the LHCB1.5 interacting protein SCO2 has been experimentally proven as chloroplastic, despite its mitochondrial prediction by ARAMEMNON (http://aramemnon.botanik.uni-koeln.de) (Table 1). SCO2s interaction with LHCB1.5 has been shown by both yeast two-hybrid and bimolecular fluorescence complementation (BiFC) method, in planta.17 Thylakoid formation requires SCO2 proteins, and loss of function mutants (sco2) show accumulation of vesicles close to the inner envelope membrane.

It is well known that several of the LHC proteins use the post-translational SRP pathway to target thylakoid membranes, which requires GTP and the cpSRP54, cpSRP43, cpFtsY, and Alb3 proteins in vitro.18,19 However, both homozygous single and double mutants with a disrupted SRP pathway remain viable in vivo, indicating at least one other available pathway is providing transport opportunities.20-22 In contrast to LHC proteins, SCO2 does not interact with cpSRP54 or cpFtsY, and it was hypothesized that some LHC proteins target thylakoids by vesicles, hence possibly explaining the previously showed redundancy.15,17,20,21

The correlation of chlorophyll and LHC proteins has been demonstrated repeatedly, showing the need of chlorophyll for LHC protein distribution to thylakoids.23 When dark grown cells of Chlamydomonas reinhardtii with impaired chlorophyll synthesis were exposed to 6 hours of light, the LHC proteins remained close to the envelope. However, when the procedure was repeated using cells with functional chlorophyll synthesis, the LHC proteins were distributed throughout the chloroplast, including thylakoid membranes.23,24 Notably, it was further found that dark grown cells accumulated vesicles after only a few minutes of light exposure,25 implying that the proliferation of vesicles could coincide with the relocation of LHC proteins in C. reinhardtii.

Previously, the Vesicle-Inducing Protein in Plastids 1 (VIPP1) has been found to be important for vesicle transport. VIPP1 knockdown mutants (vipp1) had pale green leafs, disturbed photosynthesis, lacked formation of vesicles and had inhibited thylakoid formation.26 In a follow-up study, fusion protein of VIPP1 and Protein A from Staphylococcus aureus (VIPP1-PROTEINA) was used to complement vipp1. The plants with a homozygous insertion of VIPP1-PROTEINA in vipp1 (named 2K2) had close to wild-type levels of VIPP1 protein, whereas a heterozygous insertion (named K2) generated VIPP1 levels at about 40% of the levels detected in wild-type plants. 2K2 plants had chloroplasts indistinguishable to wild type, with well-developed thylakoid membrane systems. However, the K2 plants strangely had chloroplasts ranging from only partially to fully developed, even within the same cell.27 When analyzing the levels of photosynthetic proteins, both the vipp1 mutant and K2 showed much reduced levels, but interestingly levels of LHCB1 still remained rather high for a mutant (partially) lacking thylakoid membranes. Spectroscopic analyses further indicated presence of free LHC proteins, not attached to the PSII core complex, in the vipp1 mutant plants.27 Why LHCB1 is not further down regulated remains an enigma. If LHCB1 is partly transported by vesicles and these are absent in the mutants this could explain the levels of free LHC proteins. However, this explanation currently remains a hypothesis, but reports of proteins altering the thylakoid membrane formation and organization are indeed accumulating.28

To summarize, mutants of CPRabA5e, CURT1A and C, THF1 and SCO2 all show accumulation of vesicles, indicating their direct/indirect involvement in fusion of vesicles to thylakoid membranes. The VIPP1 mutant, on the other hand, is unable to form vesicles, indicating direct/indirect involvement in budding events. Together the reports put a spotlight on the previously suggested hypothesis of an alternative pathway for LHC proteins. Are LHC proteins, and LHCB1 in particular, partly transported by vesicles in chloroplast to reach the thylakoid membranes? Our findings of CPRabA5e's indicated interaction with CURT1A and LHCB1.5, and in turn SCO2, rather supports than rejects that hypothesis. But the precise mechanism for vesicle transport and possible cargo is still unknown, and it is evident that there is a future need for experiments focusing on protein-protein interactions to unravel this.

No potential conflicts of interest were disclosed.