Fig. 2: Pit2 competes with Pit1 for binding to Pit1 to form a stable heterocomplex mainly in the cytosol.

A In vivo self-association of full-length Pit1 and Pit2. The indicated combinations of tagged proteins were transiently expressed in N. benthamiana. Co-IP was performed using anti-HA magnetic beads, and the proteins were detected by Western blot with relevant antibodies. Ponceau staining of Rubisco serves as a loading control. B In vitro pull-down assay of homo and heterocomplexes of Pit1 CC and Pit2 CC. GST, Pit1 CC-GST, or Pit2 CC-GST immobilized on GST beads was incubated with Pit1 CC-SUMO or Pit2 CC-SUMO. Input and pull-down proteins were detected by Western blot with anti-GST and anti-SUMO antibodies. C Pit2 competes with Pit1 to form heteromers in vivo. Pit1-GFP was transiently co-expressed with Pit1-HA in the presence or absence of Pit2-Myc in N. benthamiana. Co-IP was performed using anti-GFP agarose beads, and the proteins were detected by Western blot with relevant antibodies. Ponceau staining of Rubisco served as a loading control. D Pit2 CC competes with Pit1 CC to form a heteromer in vitro. Pit1 CC-GST and Pit1 CC-SUMO were co-incubated with anti-GST agarose beads and different amounts of Pit2 CC-Myc. Pull-down assay was carried out using anti-GST beads and proteins were detected by Western blot with corresponding antibodies. E Localization of Pit1 and Pit2 in rice protoplasts. Protoplasts were transfected with the fluorescent constructs Pit2-Venus with combinations of FLS2-mCherry, mCherry, or mCerulean-NLS. N, nucleus. BF, Bright-field. Scale bars, 5 μm. F Colocalization of Pit1 and Pit2 in rice protoplasts. Pit1-Venus was co-transfected with Pit2-mCherry. Scale bars, 5 μm. G Cell fractionation assay of Pit1 and Pit2. Pit1-HA, Pit2-Myc, or both were transiently expressed in N. benthamiana. Western blot was performed with indicated antibodies. T, S, and M indicate total extract, soluble fraction, and microsomal fraction, respectively. M(3X) indicates three-fold enrichment relative to T and S. Ponceau staining of Rubisco serves as a loading control. All images are representative of results repeated three times with similar results. Source data are provided as a Source Data file.