Figure 3. Subcellular localization of Ynd1 proteins and E4orf4.

A. ynd1Δ yeast cells expressing the indicated Ynd1 constructs were subjected to subcellular fractionation by differential centrifugation, and the presence of Ynd1 proteins in the various fractions was determined by a Western blot analysis. Rpb4 is a RNA polymerase II subunit which shuttles between the nucleus and the cytosol, and served here as a cytosolic fractionation marker. H: heavy membranes. L: light membranes. C: cytosol. B. ynd1Δ yeast cell extracts expressing vector control or WT Ynd1 together with E4orf4 were subjected to subcellular fractionation by differential centrifugation. Western blots were stained sequentially with antibodies recognizing tagged Ynd1, E4orf4, and the Tpd3 subunit of PP2A. C. Crude membranes were prepared from yeast cells expressing the indicated Ynd1 constructs or E4orf4, and were subjected to a Proteinase K protection assay in the presence or absence of Triton X-100. Proteins were analyzed by Western blots stained with the indicated antibodies. The Myc tag is fused to the amino terminus of Ynd1 constructs and the HA tag is fused to the cytosolic carboxyl tail. In this experiment, a longer exposure of the 518-Ynd1-containing blot allowed the presentation of a stronger signal in the blot to confirm that most of the protein was degraded by Proteinase K.