Transient transfections were carried out using FuGENE™ 6 (Roche). Stable cell lines were produced by selection for G418 resistance. The GPI-GFP construct was from S. Lacey (Southwestern University, TX), GPI-yellow fluorescent protein (YFP) was from M. Edidin (Johns Hopkins University, Baltimore, MD) Plasmids expressing VSV-Gts045-CFP and GalT-CFP were derived, by exchange of GFP for cyan fluorescent protein (CFP), from constructs described previously (Presley et al. 1997; Zaal et al. 1999). The CD59-GFP–expressing plasmid was produced by inserting CD59 cDNA between the EcoRI and BglII sites of plasmid ss-GFP (Nehls et al. 2000). Rab5S34N and Q79L were created by PCR site–directed mutagenesis and cloned KpnI/BamHI into pEGFPc1 and appropriate spectral variants (CLONTECH Laboratories, Inc.). The plasmid expressing dominant negative epsin mutant (just the DPW domain of epsin) was from P. DeCamilli (Yale University, New Haven, CT; Chen et al. 1998). Plasmid-expressing Eps15 mutant (D95/295) was from A. Benmerah (Institut Pasteur, Paris, France; Benmerah et al. 1999)