Fig. 4. The N-terminal region binds to the late Golgi/endosomal membranes. (A) YFP93 (mon2) cells expressing Mon2[A-F]p-GFP/(pFP45), Mon2[A-E]p-GFP/(pOD123), Mon2[A-B]p-GFP/(pOD125), Mon2[A]p-GFP/(pFP46), Mon2[d-F]p-GFP/(pOD122), Mon2[A-C]p/(pOD124) and Mon2[A-d]p/(pFP46) proteins were grown to mid-logarithmic phase at 30°C and processed for fluorescence microscopy. MON2[A-d] was subcloned into a centromeric (CEN) plasmid (pOD130) to reduce protein expression level and the fluorescence was detected with a confocal microscope (Nipkow). The observed localization pattern is indicated below each image. (B) For sucrose density gradients, YFP93 cells transformed with pRS316KEX2-HA and pFP45, YFP93 cells transformed with pOD123, pOD124, pOD126 or pOD127 and ODY318 (arl1::kanMX4) cells containing pFP45 were grown to mid-logarithmic phase at 30°C and subjected to equilibrium sedimentation through a sucrose gradient. Fractions were collected from the top and analyzed by western blotting using anti-HA, anti-Pep12p and anti-GFP antibodies. The first three proteins, indicated by a vertical bar, were detected from the same sucrose gradient.