Figure 1.
A high-content screen to uncover components and regulators of the vCLAMP contact site. (A) Schematic representation of the split-vCLAMP reporter strain. The strain contains the VC fragment of the split-Venus fused to the vacuolar transporter Zrc1 and the VN fragment fused to the outer mitochondrial transmembrane protein Tom70. The two fragments of the Venus protein can come into contact only when the two membranes are in very close apposition, as in a contact site. Reconstitution of the Venus protein emits a fluorescent signal, reporting on the contact site localization. (B) Schematic representation of the high-content microscopy screen. Yeast strains carrying the split-vCLAMP reporter were mated with a collection of strains each expressing one protein tagged with an mCherry fluorophore and under the strong TEF2 promoter. Haploid cells carrying the reporter and an overexpressed protein tagged with mCherry were analyzed by automated fluorescence microscopy and manually inspected for colocalization or changes in the abundance or morphology of the contact site. (C) Representative fluorescence microscopy images of the different categories of hits identified in the screen. All hits for each category are listed on the left. Images for each category correlate to the protein in bold. Scale bar represents 5 µm. (D) Representative fluorescence microscopy image of the colocalization between mCherry-Cvm1 expressed under the control of the TEF2 promoter and the split-vCLAMP reporter fluorescence, explained in A. Scale bar represents 5 µm. (E) The colocalization of Cvm1 with the split-vCLAMP signal occurs in the proximity of the vacuole and the mitochondrial network. Fluorescence microscopy analysis of mCherry-Cvm1 under the control of the TEF2 promoter, the split-vCLAMP reporter signal, Shm1-Halo stained with JF646 as a mitochondrial marker, and CMAC as vacuolar marker. Scale bar represents 5 µm. BF = Brightfield. (F) Diagram of the Cvm1 protein depicting the region predicted as homologous to α-β hydrolase fold proteins and the region predicted as intrinsically disordered.

A high-content screen to uncover components and regulators of the vCLAMP contact site. (A) Schematic representation of the split-vCLAMP reporter strain. The strain contains the VC fragment of the split-Venus fused to the vacuolar transporter Zrc1 and the VN fragment fused to the outer mitochondrial transmembrane protein Tom70. The two fragments of the Venus protein can come into contact only when the two membranes are in very close apposition, as in a contact site. Reconstitution of the Venus protein emits a fluorescent signal, reporting on the contact site localization. (B) Schematic representation of the high-content microscopy screen. Yeast strains carrying the split-vCLAMP reporter were mated with a collection of strains each expressing one protein tagged with an mCherry fluorophore and under the strong TEF2 promoter. Haploid cells carrying the reporter and an overexpressed protein tagged with mCherry were analyzed by automated fluorescence microscopy and manually inspected for colocalization or changes in the abundance or morphology of the contact site. (C) Representative fluorescence microscopy images of the different categories of hits identified in the screen. All hits for each category are listed on the left. Images for each category correlate to the protein in bold. Scale bar represents 5 µm. (D) Representative fluorescence microscopy image of the colocalization between mCherry-Cvm1 expressed under the control of the TEF2 promoter and the split-vCLAMP reporter fluorescence, explained in A. Scale bar represents 5 µm. (E) The colocalization of Cvm1 with the split-vCLAMP signal occurs in the proximity of the vacuole and the mitochondrial network. Fluorescence microscopy analysis of mCherry-Cvm1 under the control of the TEF2 promoter, the split-vCLAMP reporter signal, Shm1-Halo stained with JF646 as a mitochondrial marker, and CMAC as vacuolar marker. Scale bar represents 5 µm. BF = Brightfield. (F) Diagram of the Cvm1 protein depicting the region predicted as homologous to α-β hydrolase fold proteins and the region predicted as intrinsically disordered.

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