Skip to main content
. 2013 Jun 20;8(6):e67307. doi: 10.1371/journal.pone.0067307

Figure 2. Localization of CORVET requires the N-terminal domains of Vps3 or Vps8.

Figure 2

(A) Localization of N-terminally truncated Vps3 and Vps8. Vps3 and Vps8 were genomically tagged with 3xmCherry at their C-temini, and colocalized with genomically tagged GFP-Pep12. Size bar, 10 µm. (B) Colocalization of Vps8 with Vps3. Analysis was performed as in A. Vps3 was genomically tagged with 3xmCherry at the C-terminus, whereas Vps8 was tagged with yeGFP. Size bar, 10 µm. (C) Subcellular localization of dually truncated CORVET. TAP-tagged Vps8 was monitored in wild-type and in cells expressing Vps3ΔN and truncated Vps8. Cells (Total, T) were fractionated to obtain a pellet (P100) and supernatant (S100) after the final centrifugation at 100,000 g. Western blots were decorated against the TAP tag to identify Vps8. Decoration with antibodies against Arc1 and Vac8 was used as control for cytosolic and membrane-enriched fractions. (D) Endocytosis of Ste3 in cells expressing truncated Vps3 and Vps8. Endocytosis in the respective strains was followed by fluorescence microscopy of C-terminally GFP-tagged Ste3, expressed from 2µ-plasmids. To monitor vacuole morphology in parallel, cells were treated with FM4–64 beforehand. Size bar, 10 µm.