Fig. 1: Isolation of deip1 mutants, genetic complementation, subcellular localization analysis of the DEIP1 protein, and thylakoid protein accumulation in wild-type and mutant plants.

a Mutant phenotypes. 7-day-old seedlings of the wild type (WT), the deip1-1, deip1-2, and deip1-3 mutants, and the four complemented lines deip1-C1-4 were raised under a moderate light intensity of 95 µmol m⁻² s⁻¹. Scale bars: 5 mm. n = 6 independent biological replicates. b DEIP1 transcript accumulation in the Arabidopsis deip1-1, deip1-2, and deip1-3 mutants relative to wild-type plants (WT). The qRT-PCR data for the mutants were normalized and compared to the wild type. n = 3 biological replicates; error bars indicate standard deviation; Statistical significance was determined by one-way ANOVA, P values were adjusted by Tukey post-test for multiple comparisons, ***P < 0.001. c Localization of the DEIP1-GFP fusion protein as analyzed by confocal laser-scanning microscopy. The green fluorescence of DEIP1-GFP (green; upper panels), the chlorophyll fluorescence (red; middle), and the merged images (bottom) are shown. Scale bars: 50 μm (left panels), 20 μm (middle panels) and 5 μm (right panels). n = 3 independent biological replicates. d Immunoblot analysis of DEIP1-GFP localization in complemented line deip1-C4. Samples of 10 µg total chloroplast protein (C), and purified stroma (S) and thylakoid fractions were separated in a 12.5% PAA gel, and probed with anti-GFP, anti-RbcL, and anti-LCHB2 antibodies. n = 3 independent biological replicates. e Localization of DEIP1-GFP in intact thylakoids (T), and in fractionated thylakoids separated into grana core (G.C), grana margins (G.M), and stroma lamellae (S.L). Protein amounts equivalent to 1 µg chlorophyll were loaded for each fraction, and the blots were hybridized to an anti-GFP antibody for detection of the DEIP1-GFP fusion protein. The quality of the fractionation was assessed with antibodies against PsbA (highly abundant in G.C), PsaB (highly abundant in S.L), PetA (primarily present in G.M, but also in G.C and S.L), AtpB (primarily present in GM and SL), and CURT1A (highly abundant in GM). The Coomassie-stained PAA gel (CBB) is shown below the immunoblot. n = 3 independent biological replicates. f Salt wash of thylakoid samples from deip1-C4. Thylakoid samples equal to 1 µg of chlorophyll were incubated with 1 M NaCl for 1 h and separated in pellet (P) and soluble (S) fractions by centrifugation. Proteins present in the pellet and soluble fractions were resolved in a 12% PAA gel, and probed with anti-GFP, anti-AtpB, and anti-LHCB2 antibodies. n = 3 independent biological replicates. g Trypsin protection assay. Thylakoids were isolated from deip1-C4, protein samples equivalent to 2 µg of chlorophyll were incubated with 1 µg mL⁻¹ trypsin at 37 °C for 30 min, resolved in a 12.5% PAA gel, and probed with anti-GFP, anti-PSBO (detecting a marker protein that resides at the lumenal side of the membrane and, therefore, is protected from tryptic digestion), and anti-PsaB antibodies (recognizing a protein containing domains exposed to the stromal side and, therefore, being susceptible to tryptic digestion). The DEIP1-GFP degradation products resulting from tryptic digestion are indicated as DEIP1-GFP*. As a control for equal loading, the Coomassie-stained PAA gel (CBB) is shown below the immunoblot. The cartoon illustrates the topology of the DEIP1-GFP protein in the thylakoid membrane. n = 2 independent biological replicates.