Fig. 3. Mon2p is associated with the late Golgi and early endosomes. (A) Mon2p-GFP colocalizes with Kex2p-HA and Pep12p on a sucrose density gradient. JEY003 (MON2-GFP) cells harboring pRS316-KEX2-HA were grown to mid-logarithmic phase at 30°C and subsequently lysed. The P100 fraction was prepared and subjected to equilibrium sedimentation through a sucrose gradient. Fractions were collected from the top, subjected to SDS-PAGE and analyzed by western blotting using anti-GFP, anti-HA, anti-Mnn1p and antiPep12p antibodies. (B) Mon2p-GFP partially colocalizes with late-Golgi markers (Sec7pDsRed and PH_FAPP1-DsRed), but shows no overlap with a late endosomal marker (DsRedFYVE_EEA1). Strains chromosomally expressing Mon2p-GFP and harboring the indicated fusion construct on a plasmid were grown to mid-logarithmic phase and observed by fluorescence microscopy as described in Materials and Methods. Images are representative of more than 100 cells observed.