Fig. 5. Membrane association of wild-type and variant Ugt51 proteins. Pichia pastoris ugt51Δ cells expressing different GFP-tagged Ugt51 fusion proteins were transferred from methanol to glucose medium and harvested after 1 h. The cells were lysed and the homogenates were fractionated by centrifugation at 100 000 g. The pellets (P1) and supernatant fractions (S1) were subjected to immunoblot analysis using anti-GFP antibody. In addition, the pellet fraction was treated with 1% Triton X-100 (TX-100), 1 M KCl; or 0.1 M Na2CO3, pH 11.0. After subsequent re-centrifugation, the resulting supernatants (S) and pellets (P) were also subjected to immunoblot analysis. Note that only Ugt51 lacking the PH domain was relocalized to the soluble fraction following Triton X-100 treatment. On the other hand, Paz16-CFP, another Paz protein, could be extracted to the soluble fraction by treatment with 1% Triton X-100 or 1 M KCl.