Plasma membrane (PM) purity verification and PM-related proteins evaluation. a Western blot examination of plasma membrane enrichment. Proteins from entire cell (EC) lysates, microsomal vesicles (MSV), PM (plasma membrane vesicles) and carbonate-washed PM (CPM) were separated by 10% SDS-PAGE, transferred to PVDF membranes and detected with antibodies for PMA2, a PM marker; COXII, a mitochondrial marker; V-ATPase, a vacuole marker; S14-1, a ribosome marker; or H1, a nucleus marker. For detection of PMA2, COXII and V-ATPase, 5 ?g protein was loaded per lane; for detection of S14-1 and H1, 10 ?g protein was loaded per lane. b Summary of proteins with transmembrane domain (TMD) predicted with use of HMMTOP 2.0. c Venn diagram depicting the distribution of proteins with different lipid modifications. GPI, glycosylphosphatidylinositol anchor; Pre, prenylation site; Myr, myristoylation site; Pal, palmitoylation site. d Proteins predicted to have a TMD or post-translational modification (PTM) or both. e Protein subcellular locations annotated by Gene Ontology or WoLF PSORT showed that 1,121 of the 1,797 proteins with a TMD and/or PTM had PM location information

Subcellular localization of CsMTP6. (A) Mitochondrial localization of CsMTP6 in protoplasts prepared from suspensions of Arabidopsis cells: 1, GFP fluorescence of protoplast expressing CsMTP6-GFP; 2, MitoTracker red fluorescence; 3, merged image; 4, transmission image of the same protoplast. The scale bar is 10 µm. (B) Immunolocalization of CsMTP6 in cucumber root cells. SDS-PAGE followed by western blot analysis of plasma membrane (PM), tonoplast (Ton), and mitochondrial (Mit) and plastidial (Plast) fractions. The fractions (15 ?g of protein per lane) were blotted with the primary antibodies to identify marker enzymes for the plasma membrane (PM-ATPase), tonoplast (V-ATPase), plastids (Toc75), mitochondria (COXII), and to localize CsMTP6. L, protein ladder.

Effects of CsMTP6 expression on mitochondrial Fe and Mn contents and Mn-SOD activity in Arabidopsis protoplasts. (A) Western blot analysis of the purity of mitochondrial and plastidial fractions prepared from protoplasts expressing CsMTP6 using primary antibodies raised against GFP (to detect CsMTP6-GFP), and marker proteins for plastids (Toc75) and mitochondria. The mitochondria and plastid fractions were prepared from protoplasts by discontinuous density gradient centrifugation in Percoll. (B) Fe and (C) Mn content in the mitochondrial fraction prepared from Arabidopsis protoplasts expressing CsMTP6 or transformed with the empty vector. Data are means (±SD) from three separate experiments. (D) Mitochondrial Mn-SOD activity in protoplasts expressing CsMTP6. Data are means (±SD) from three separate experiments, expressed as enzyme units (U). Significant differences were determined using Student’s t-test.

The levels of CsMTP6 transcript and protein under different Fe and Mn availability. (A) Real-time analysis of CsMTP6 expression in the roots of 2-week-old cucumber seedlings growing under control Fe (75 µM), control Mn (0.1 µM), excess Fe (1 mM), excess Mn (10 µM), Fe deficiency, or Mn deficiency. Data are mean CsMTP6 transcript levels (±SD) relative to the constitutively expressed reference gene CACS calculated from the arithmetic means of ?Cp values obtained in three independent experiments. (B) Western blot analysis of CsMTP6 protein levels in mitochondria isolated from roots of cucumbers grown under different Fe and Mn treatments for 2 weeks. Immunoblots of the same mitochondria were probed with antibodies against mitochondrial COXII cytochrome oxidase to ensure equal loading of membrane protein (10 µg) onto SDS-PAGE. Significant differences were determined using Tukey’s test.

FSL0260 interacts with mitochondrial complex I and inhibits its activity. (a) Inhibition of complex I by FSL0260. Oxygen consumption rate (OCR) of isolated mitochondria from potato tuber was monitored with deamino-NADH in the absence or presence of FSL0260. (b) Inhibition of deamino-NADH oxidation by FSL0260. Deamino-NADH oxidation was measured by spectrometry using sonicated mitochondria isolated from the potato tuber. The experiment was conducted using three biological replicates. Error bars represent the mean ± SE (n?=?3). (c) Pull-down assay of FLS0260. Sonicated potato mitochondria were incubated with control or FSL0260 beads. Immunoblot assay was performed using an anti-gamma CA antibody and an anti-COX II antibody.