A) Schematic representation of Sna3 depicting its two predicted transmembrane domains and the relative location of its PY motif and lysine residues.
B) Localization of Sna3-GFP or Sna3ΔK-GFP in pep4Δ and pep4Δ vps4Δ cells Bar = 5 μm.
C) Post-nuclear supernatants were prepared from cells in A and treated in the absence of presence of 25 μg /ml trypsin for 15 min at 22°C before addition of protease inhibitors. Samples were then subjected to immunoblot analysis using antibodies against GFP and CPY.
D) Lysates from wild-type cells expressing versions of Sna3 (wild-type, ΔK and ΔPY) tagged with a C terminal 2 x HA epitope were prepared for iummunoblot analysis using anti-HA antibodies. High (50 μg) and low (20 μg) levels of lysates are depicted (upper panel). * indicates ubiquitinated species. An over-exposure better illustrating ubiquitinated bands is shown in lower panel.
E) Trypsin protection experiments with post-nuclear supernatants from pep4Δ cells expressing HA-tagged Sna3 and Sna3ΔK. Samples were treated with or without trypsin and in the presence or absence of Triton X-100.