Supplementary Materials Appendix EMBJ-35-1991-s001. and Sec7\HA\positive AB\like structure (Fig?EV3B), indicating the induction of autophagy and GOMED. Most structures had either Romidepsin (FK228 ,Depsipeptide) one Romidepsin (FK228 ,Depsipeptide) of these signals and only a few of the structures contained both signals (Fig?EV3C). Note that the membrane of AB\like structure was similar to that of AB (Fig?EV3B). We also examined the extent of GFP\Sft2 processing Romidepsin (FK228 ,Depsipeptide) during AmphoB treatment and found that the processing in cells were incubated at 37C (temperature shift) for 3?h, and the localization of GFP\Atg8 and Sec7\mRFP in the vacuoles was observed by confocal microscopy. The arrowhead and arrow indicate the Sec7\mRFP puncta with and without GFP\Atg8, respectively, indicating the induction of autophagy and GOMED in WT yeast cells. Scale bars?=?2?m. B, C GFP\Atg8/Sec7\HA\expressing cells were incubated at 37C (temperature shift) for 3?h, and freeze replica immunolabeling for HA (15?nm gold) and GFP (10?nm gold) was performed. There were two types of structures in the vacuoles: GFP\Atg8\positive AB and Sec7\HA\positive AB\like structure, indicating the induction of GOMED in WT yeast cells. Scale bar?=?1?m. In (C), the percentage of GFP\Atg8\positive ABs and Sec7\HA\positive AB\like structures was calculated (mean??s.e.m., for 10?min. After washing twice with 1?ml of ice\cold acetone, cells were air\dried and suspended in 100?l of sample buffer. After disruption of the cell walls by vortexing with an equal volume of acid\washed glass beads for 3?min, proteins were boiled at 100C for 3?min. Samples were then loaded on a 15% polyacrylamide gel and electrophoresed. A standard semidry Western blot transfer procedure was performed using a PVDF membrane. After blotting, membranes were probed by incubation with anti\GFP antibody overnight at 4C. After washing the membrane twice with T\TBS for 10?min, the second antibody was applied for 1?h at room temperature. After washing membranes, the GFP signal was detected using the ECL kit. Fluorescence and phase\contrast microscopy in yeast cells For fluorescence microscopy, yeast cells transfected with GFP fusion proteins or mRFP fusion proteins were visualized with a confocal microscope (Zeiss; LSM510 system). Samples were also observed by DIC microscopy (Nikon; TE\2000), and video images were taken at 200 magnification using a CCD camera (Keyence). For phase\contrast microscopy, yeast cells were examined under an Olympus BS2 microscope with a 100 oil\immersion objective for phase\contrast optics. Images were obtained using DP2\BSW. Measurement of trafficking efficiency from Golgi to plasma membrane Yeast cells with HA(3)\tagged Hsp150 were precipitated with trichloroacetic acid for 10?min on ice. Cells were collected by centrifugation and performed Western blotting using anti\HA antibody as described in GFP processing assay. This protein is transported from the ER through the Golgi apparatus and subsequently secreted away. The ER\localized form is detected at 87?kD, whereas the Golgi\localized form is at Romidepsin (FK228 ,Depsipeptide) 150?kD in SDSCPAGE, due to the glycosylation. To examine the extent of each size of HA\Hsp150, we can estimate the trafficking efficiency from the ER to Golgi and Golgi to PM. Yeast subcellular fractionation Cells were collected and converted to spheroplasts as described previously (Sato for 15?min at 4C to yield an intermediate speed supernatant fraction, and further centrifuged at 100,000??for 60?min at 4C to obtain crude membrane fraction. To isolate vacuole and KRT17 vacuolar membrane, spheroplasts were suspended in 10 volumes of buffer A (10?mM Mes/Tris (pH 6.9), 0.1?mM MgCl2, 12% Ficoll\400) and homogenized with Dounce homogenizer. The solution was suspended in 10?ml of buffer B (10?mM MES/Tris (pH 6.9), 0.5 MgCl2, 8% Ficoll\400) and centrifuged at 51,900??for 30?min at 4C. Romidepsin (FK228 ,Depsipeptide) The white layer on top was collected and resuspended in buffer B, and.