GET3B is involved in chloroplast biogenesis and interacts with the thylakoidal ALB3 and ALB4 insertases

Abstract

Proteomic, functional physiological analyses of get3b mutant plants highlight GET3B's role in chloroplast function. Genetic and interaction analyses indicate get3b and srp54 as mutual potentiators that might share terminal insertases. Protein targeting and insertion into membranes are essential for cellular organization and organelle function. The Guided Entry of Tail-anchored (GET) pathway facilitates the post-translational targeting and insertion of tail-anchored (TA) membrane proteins. Arabidopsis thaliana has four GET3 homologues, including AtGET3B and AtGET3D localized to chloroplasts. These photosynthetic organelles possess complex membrane systems, and the mechanisms underlying their protein targeting and membrane biogenesis are not fully understood. This study conducted a comprehensive proteomic analysis of get3b mutant plastids, which displayed significant alterations. Fluorometric based complex assembly as well as CO₂ assimilation analyses confirmed that disruption of GET3B function displayed a significant impact on photosystem II assembly as well as carbon fixation, respectively, indicating a functional role in chloroplast biogenesis. Additionally, genetic interactions were found between GET3B and the two component STIC system, which cooperates with the cpSRP pathway which is involved in the co-translational sorting of thylakoid proteins. Further, physical interactions were observed between GET3B and the C-terminus of ALB3 and ALB4 in vitro and the full length proteins in vivo, indicating a role of GET3B in protein targeting and membrane integration within chloroplasts. These findings enhance our understanding of GET3B's involvement in stromal protein targeting and thylakoidal biogenesis.

Keywords: ALB3; ALB4; GET3B; STIC1; STIC2; cpSRP54.

Fig. 1

The get3b mutation and the downstream effects on the chloroplast proteome. A Gene model of GET3B and the corresponding T-DNA insertion line analyzed in this study. B Genotyping via PCR of get3b. The locations of the used primers are indicated in A as arrowheads. C Semi RT-PCR to validate the missing transcript. D Western blot analysis of total protein lysates. Wild-type and get3b plastids were isolated and subjected to LC-MS/MS followed by label free quantification. E Statistical analysis of proteinaceous hits that were significantly effected in get3b chloroplasts (see Supplemental Table S1 for more details). The significantly depleted (F) and significantly enriched (G) proteinaceous hits were categorized according to their cellular function. The color coding in E corresponds to those in F and G

Fig. 2

GET3B function is involved in the assembly of photosystem (PS) II. A Seedlings were germinated and cultivated under etiolating conditions for 5 days on 0% sucrose (top) or 1% sucrose (bottom) for 5 days before being normally illuminated and subjected to pulse amplitude modulation (PAM) measurements. The Fv/Fm ratio (maximum quantum yield) was determined after 1, 7, 24 hours to monitor PS II assembly. B Seedlings were grown under normal illumination on 0% sucrose (top) and 1% sucrose (bottom). After 5 days these were subjected to PAM measurements. C Seedlings were cultivated under normal conditions for 4 days and either subjected to PAM measurements or (D) used for chlorophyll isolation. Statistical analyses for (A) and (B) were performed using the Student’s t-test, for three independent experiments with 40 seedlings per experiment. Statistical analyses for (C) were performed with ANOVA for 15 independent experiments with 40 seedlings per experiment as well as (D) for 3 experiments with 120 seedlings per experiment

Fig. 3

GET3B function is involved in CO₂ assimilation. 4-week-old plants of the depicted lines were subjected to infrared gas analyzer (IRGA) measurements. Data points represent the mean of 4 independent measurements. Asterisks denote significant differences (one way anova ***P < 0.001). N.S. : not significant

Fig. 4

Genetic interactions are seen when mutations in GET3B are combined with mutations affecting ALB4 and/or STIC2. Plants were grown in 12-h light/12-h dark conditions for 4 weeks. Top row: Images of A stic2, B alb4 C srp54, D Columbia wild-type plants. Middle row: Phenotypes when get3b mutations were introduced: E stic2 get3b, F alb4 get3b, G srp54 get3b, H get3b plants. Bottom row: I alb4 stic2, J alb4 stic2 get3b, K alb4 srp54, L alb4 srp54 get3b plants. Black areas were added so that plants would be shown at equal magnification. Bars= 1 cm. M Rosette diameters of plants with different genotypes in the absence (left) or presence (right) of srp54 mutations. Data are presented as means ± SE (n = 2–10 individual plants). Asterisks designate significant differences (Student’s t test, * P < 0.05, ** P < 0.01) from wild-type or between the samples indicated with brackets

Fig. 5

Transgenes encoding wild-type GET3B can complement alb4 srp54 get3b triple mutants, but not transgenes encoding GET3B with a D124N mutation. Plants were grown in 12-h light/12-h dark conditions for 4 weeks. Top row: Images of (A) alb4 srp54 plants, and (B, C) two independent lines of transgenic plants carrying the ProUBI10:GET3B-STREP transgene in an alb4 srp54 get3b background. Bottom row: Images of (D) alb4 srp54 get3b plants, and E, F two independent lines of transgenic plants carrying the ProUBI10:GET3B(D124N)-STREP transgene in an alb4 srp54 get3b background. Bars= 1 cm. G Rosette diameters of alb4 srp54 plants and three independent lines of alb4 srp54 get3b plants carrying the ProUBI10:GET3B-STREP transgene. H Rosette diameters of untransformed (T0) alb4 srp54 get3b plants and four independent lines of alb4 srp54 get3b plants carrying the ProUBI10:GET3B(D124N)-STREP transgene. Data are presented as means + SE (n = 8–10 individual plants in G, n = 3–8 individual plants in H). Asterisks designate significant differences (Student’s t test, *P < 0.05, **P < 0.01) between the transgenic plants and either alb4 srp54 plants (G) or alb4 srp54 get3b plants (H)

Fig. 6

Expression of transgenes encoding GET3B with a D124N mutation leads to reductions in plant size in alb4 or stic2 genetic backgrounds. Plants were grown in 12-h light/12-h dark conditions for 4 weeks. Top row: Images of (A) Columbia wild-type, (B) alb4, and (C) stic2 plants. Bottom row: Transgenic plants carrying the (D) ProUBI10:GET3B-STREP transgene in a stic2 background, (E) ProUBI10:GET3B(D124N)-STREP transgene in an alb4 background and (F) ProUBI10:GET3B(D124N)-STREP transgene in a stic2 background. Bars= 1 cm. G Rosette diameters of untransformed (T0) stic2 plants and two independent lines of transgenic stic2 plants carrying the ProUBI10:GET3B-STREP transgene. H Rosette diameters of untransformed (T0) alb4 (left) or stic2 (right) plants and two independent lines each of transgenic alb4 or stic2 plants carrying the ProUBI10:GET3B(D124N)-STREP transgene. Data are presented as means + SE (n = 10–14 individual plants in G, n = 10–15 individual plants in H). Asterisks indicate significant differences (Student’s t test, *P < 0.05, **P < 0.01) between the transgenic plants and untransformed (T0) plants of the same genotype

Fig. 7

Get3b interacts with the thylakoid membrane proteins Alb3 and Alb4. A In vitro pull-down assays were performed with recombinant GST-tagged mature Get3b (GST-Get3b) and the His-tagged C-terminus of Alb3 (Alb3C-His) or Alb4 (Alb4C-His). Samples containing GST and the C-termini, or the C-termini alone served as negative controls. Approximately 4% of the load samples were applied to SDS-PAGE analysis and stained with Coomassie. B 10% of the eluates of the pull-down assay were subjected to immunoblot analysis using antibodies specific for Alb3, Alb4, or GST. The asterisks indicate truncated forms of the GST-Get3b. The closed circle indicates a nonspecific reaction of the anti-Alb4 antibody with GST. C Split-ubiquitin yeast two-hybrid assay using mature Get3b and mature full-length Alb3. Alb3 was used as fusion with the C-terminal half of ubiquitin (Cub) at its C-terminus (Alb3-Cub). Get3b was used as fusion with a modified N-terminal half of ubiquitin (NubG) at its N-terminus (NubG-Get3b). The unrelated endoplasmic reticulum membrane protein Alg5 fused to wild-type Nub (Alg5-NubI) and NubG (Alg5-NubG) served as positive and negative controls for the Cub-fusion construct, respectively. DSY-1 yeast colonies were first plated on permissive medium (−LT, lacking Leu and Trp) and on selective medium (−LTH, lacking Leu, Trp, and His). D Same assay as in (C) but using mature full-length Alb4 as bait. E Split-ubiquitin interaction tests of SecY1-Cub with NubG-Get3b and of Alg5-Cub with NubG-Get3b as negative control