Panel A: A schematic representation of Model 1, illustrating the Vps13-Mcp1 complex driving lipid exchange at membrane contact sites bridged by ChiMERA. Key structural components labeled include the ER, OMM, ChiMERA, Mcp1, and Vps13. Panel B: Representative micrographs demonstrating the recruitment of Vps13 superscript GFP to Mdm34-mCherry foci following Mcp1 overexpression. Visualization is supported by mtBFP and Vps13 superscript GFP channel labeling. Panel C: A bar graph comparing the predicted versus observed colocalization of Vps13 superscript GFP foci with Mdm34-mCherry under Mcp1 overexpression conditions. Error bars represent the standard error of the mean (SEM). Panel D: Analysis of progeny viability via representative tetrads from MDM12/mdm12 delta VPS13/vps13 delta diploids harboring ChiMERA. Genotypes are labeled to distinguish wild-type, single mutants (mdm12 delta, vps13 delta), and the mdm12 delta vps13 delta plus ChiMERA rescue. Panel E: Fluorescence micrographs detailing Vps13 superscript GFP localization relative to mitochondria (mtBFP) and peroxisomes (mCherry-SKL) during Mcp1 overexpression. Comparisons are shown in the presence and absence of Gem1, with channels labeled for organelle markers and Vps13/peroxisome overlap. Panel F: Schematic representation of Model 2, illustrating the functional redundancy between Mdm12 and Mdm34 within ChiMERA-stabilized ER-mitochondria interfaces. Labeled components include the ER, OMM, ChiMERA, and the ERMES subunits (Mmm1, Mdm34, Mdm12, Mdm10). Panel G: Growth phenotypes assessed by spot assay for mdm12 delta, mdm34 delta, and the mdm12 delta, mdm34 delta double mutant, comparing strains with and without ChiMERA expression.
Vps13 and joint loss of Mdm12-Mdm34 are not required for ERMES rescue by ChiMERA. (A) Schematic showing the model in which Vps13-Mcp1 can transfer lipids at ChiMERA-induced ER–mitochondria contact sites. (B) Representative images of Vps13^GFP colocalizing with Mdm34-mCherry upon Mcp1 overexpression. (C) Quantification of the proportion of Mcp1 overexpression-dependent Vps13^GFP foci expected and observed to colocalize with Mdm34-mCherry. Statistical significance was quantified with a chi-squared test. The expected value is probability a Vps13^GFP foci would colocalize by random chance. Error bars indicate ± standard error of the mean. Statistical N is three independent experiments. **** denotes P < 0.0001. (D) Representative tetrads from the sporulation of MDM12/mdm12Δ VPS13/vps13Δ diploids expressing ChiMERA. (E) Representative images of Vps13^GFP subcellular localization in comparison with mitochondria (mtBFP) and peroxisomes (mCherry-SKL) in Mcp1 overexpression conditions, both with and without Gem1. (F) Schematic of Model 2, whereby Mdm12 and Mdm34 are functionally redundant with one another upon ChiMERA-induced tethering of the ER to the mitochondria. (G) Representative spot assay of mdm12Δ, mdm34Δ, and mdm12Δmdm34Δ both with and without ChiMERA expression. Scale bars are 2 μm.