FIGURE 1:

Artificial tethers cannot compensate for the lack of vCLAMP. (A) Generation of artificial vCLAMP tethers. Cells expressing different vacuolar membrane proteins C-terminally tagged with GFP were observed by fluorescence microscopy. Where indicated, the attachment of these proteins to mitochondria was induced by tagging Tom70 C-terminally with a nanobody that recognized GFP. Shm1–3xmCherry is used to visualize mitochondria and CMAC staining to label the vacuole lumen. The scale bar represents 2 μm. (B) vCLAMP impaired cells display a growth defect in the presence of zinc or iron. Wild type, vps39Δ, or vps39^12xM strains were grown as serial dilutions on YPD, YPD containing 3 mM ZnCl₂, and YPD containing 10 mM Fe(NH₄)SO₄. (C) Artificial tethers do not suppress the growth defects of vCLAMP-impaired strains. Strains containing the vCLAMP-impaired vps39^12xM allele with or without the artificial tethers, as shown in A, were grown as serial dilutions on YPD, YPD containing 3 mM ZnCl₂, and YPD containing 10 mM Fe(NH₄)SO_4.