ER-mitochondrial junctions can be bypassed by dominant mutations in the endosomal protein Vps13

Abstract

The endoplasmic reticulum-mitochondria encounter structure (ERMES) complex tethers the endoplasmic reticulum and the mitochondria. It is thought to facilitate interorganelle lipid exchange and influence mitochondrial dynamics and mitochondrial DNA maintenance. Despite this important role, ERMES is not found in metazoans. Here, we identified single amino acid substitutions in Vps13 (vacuolar protein sorting 13), a large universally conserved eukaryotic protein, which suppress all measured phenotypic consequences of ERMES deficiency. Combined loss of VPS13 and ERMES is lethal, indicating that Vps13 and ERMES function in redundant pathways. Vps13 dynamically localizes to vacuole-mitochondria and to vacuole-nucleus contact sites depending on growth conditions, suggesting that ERMES function can be bypassed by the activity of other contact sites, and that contact sites establish a growth condition-regulated organelle network.

Figure 1.

A dominant mutation in VPS13 suppresses the growth defect of ERMES mutants. (A) Serial dilutions of strains of the indicated genotypes on fermentable (YPD: YP + 2% dextrose) or nonfermentable (YPEG: YP + 3% glycerol + 1.5% ethanol) media. Top: WT. Middle: isogenic mmm1Δ strain from a published deletion library (Giaever et al., 2002) that bears a suppressor mutation (SUP⁺). Bottom: isogenic mmm1Δ strain without suppressor mutation (SUP⁻). (B) Tetrad analysis of an MMM1/mmm1Δ heterozygote (left), a MMM1/mmm1Δ; SUP⁻/SUP⁺ heterozygote (middle), and a mmm1Δ homozygote, SUP⁻/SUP⁺ heterozygote (right). (C) Quality scores of SNPs were classified in three categories: SNPs found into both the SUP⁺ and SUP⁻ DNA pools (left), SNPs found in the SUP⁻ pool only (middle), and SNPs found in the SUP⁺ pool (right). Low-scoring SNPs represent sequencing errors while high scoring ones represent bona fide variants. (D) A diploid MMM1/mmm1Δ heterozygote was transformed with a plasmid encoding WT Vps13 (pVPS13, left) or the D716H allele (pVPS13(D716H), right). Spores circled in red bear both the deletion allele and the indicated plasmid. (E) A CEN/ARS plasmid bearing the VPS13(L1627S) allele (pVPS13(L1627S)) or its WT counterpart (pVPS13) were transformed into mmm1Δ and mdm10Δ cells. Transformants were spotted and streaked on YPD.

Figure 2.

The VPS13(D716H) allele suppresses pleiotropic consequences of ERMES deficiency. (A) Images of cells of the indicated genotype bearing GFP-tagged MDM34 (green) and a mitochondrial marker (mtDsRed, magenta). ERMES foci are indicated (arrowheads). The cell outline is shown in blue. Bar, 2 µm. (B) Cells of the indicated genotype were imaged as in A. A shape quotient was measured for each mitochondria. The number of mitochondria analyzed is indicated above each graph. (C) Cells of the indicated genotypes stained with DAPI. DAPI stains both nuclear DNA (solid arrowheads) and mtDNA (open arrowheads). Bar, 2 µm. (D) The number of cells in C displaying mitochondrial DAPI staining (with mtDNA) or not (no mtDNA) was counted for each genotype. The number of cells analyzed is indicated above. P < 10⁻¹⁰⁰ for a t test and a Fisher’s exact test to compare mmm1Δ VPS13 and mmm1Δ VPS13(D716H) in B and D, respectively.

Figure 3.

Vps13 localizes to alternative membrane contact sites. (A) Assessment of the functionality of GFP-tagged Vps13 using synthetic lethality with MMM1. mmm1 thermo-sensitive strains harboring a GFP inserted in Vps13 after amino acid 499 (1), 446 (3), 473 (4), or untagged Vps13 (2) were streaked onto YPD and grown at the nonpermissive temperature (37°C). Only GFP inserted after amino acid 499 yields a functional protein. (B) Intracellular localization of Vps13^GFP in otherwise WT cells grown on SC-Dextrose, expressing a mitochondrial marker (mtBFP) and stained with a vacuole dye (FM4-64). Vps13^GFP is often found in foci colocalizing with the vacuole and mitochondria. Bar, 2 µm. (C) Intracellular localization of Vps13^GFP in otherwise Sec61-mCherry cells grown on SC-Glycerol, and stained with the vacuole marker CellTracker Blue CMAC. Vps13^GFP relocalizes to NVJs (yellow arrowheads). (D) Quantification of the number of cells in which Vps13^GFP shows NVJ localization in dextrose- (SC-D) and glycerol- (SC-Gly) containing medium. *, P < 10⁻¹⁰⁰ from a Fisher’s exact test. (E) Time course showing the percentage of Vps13 found in NVJs upon carbon source shifting. At time 0, cells grown to exponential phase in dextrose-replete (+D) or depleted (-D) medium were washed and resuspended in indicated medium. Percentages were calculated automatically with ImageJ Script S3.

Figure 4.

Suppressor alleles of Vps13 fail to relocalize to NVJs. (A and B) Tetrad dissection of sporulated MMM1/mmm1Δ VPS13/VPS13(D716H) diploids in which the VPS13^GFP construct has been engineered in the VPS13 (A) or VPS13(D716H) (B) alleles. (C) Cells harboring the Vps13^GFP construct in the indicated VPS13 allele were grown in dextrose- (SC-D; C) or glycerol-containing (SC-gly) medium (D), and stained with DAPI (mitochondria, red) and FM4-64 (vacuole, blue). The formation of streaks (arrowheads) denotes the relocalization of Vps13^GFP to NVJs. Bar, 2 µm. (E) Quantification of the number of cells showing streaks in the indicated conditions. *, P < 10⁻²⁰; n.s., P > 0.05 from a Fisher’s exact test.