Anal Chem. involved with membrane trafficking taking place between lysosomes and endosomes. INTRODUCTION A lot of the membrane-trafficking occasions in metazoans are powered by microtubule-based molecular motors. However the increase in the number of new unconventional myosins and the recent demonstration that intracellular compartments of mammalian cells move in vivo trans-Zeatin and in vitro on actin filaments stimulated the investigation of the actin-based membrane trafficking in metazoan organisms (Langford embryo (Mermall and Miller, 1995 ). The majority of our understanding of the functional properties of myosin Is derived from studies on amoebae and yeast. Unlike the double-headed structure of myosin II or myosin V, myosin Is are single-headed, low-molecular-weight members of the myosin superfamily. Although, all myosin Is exhibit in their tail a positively charged region that has been shown to bind directly to anionic lipids, myosin Is can be divided into distinct subclasses based on sequence homologies in their head and tail domains (Coluccio and Conaty, 1993 ; Ruppert (1996a) were grown at 37C under 10% CO2 in Coons F-12 modified medium (Seromed, Berlin, Germany) supplemented with 10% FCS (Seromed) and penicillin (10 U/ml) and streptomycin (10 g/ml) (Seromed) in the case of the BWTG3 cells or supplemented with 0.7 mg/ml Geneticin, (Life Technologies, Paisley, trans-Zeatin Scotland) in the case of mock cells or the cellular clones producing BBMI or the truncated BBMI proteins. Immunoprecipitation, Immunoblotting, and Mass Spectrometry Analysis Immunoprecipitation.Cells were grown 2 d on a 10-cm Petri dish and lysed in 1 ml of 10 mM Tris, pH 7.4, containing 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, and 0.1% SDS (immunoprecipitation buffer) on ice. After centrifugation for 10 min at 10,000 (1996) . The supernatant (0.5 ml) was mixed on the target trans-Zeatin of the mass spectrometer with 0.5 ml of a saturated solution of 2,5-dihydroxybenzoic acid in 0.1% aqueous trifluoroacetic acid. Peptide molecular weights were determined by matrix-assisted laser desorption and ionizationCtime of flight analysis. Spectra were obtained in positive reflection mode on a Voyager Elite matrix-assisted laser desorption and ionizationCtime of flight mass spectrometer (Perceptive Biosystems, Framingham, MA) equipped with a delayed extraction device. The peptides maps identified with this method have been compared with the OWL, European Molecular Biology Laboratory, and Swiss data bases. Immunofluorescence Microscopy For immunofluorescence analysis cells were grown 2 d on coverslips and incubated overnight in cell culture medium containing 10 mM sodium butyrate in the case of stable cell lines producing BBMI, BBMI446, or BBMI-Tail. Internalization of Transferrin.Cells were washed three times with RPMI 1640 medium followed by a 30-min incubation period with RPMI 1640 medium at 37C. The cells were then incubated 20 min at Trp53inp1 37C with biotinylated transferrin at 20 g/ml (Sigma) in RPMI 1640 medium. Then cells were washed three times with cold RPMI 1640 medium containing 0.1 mg/ml BSA and processed for immunofluorescence analysis. Biotinylated transferrin was detected with streptavidin-conjugated with Texas Red from Molecular Probes (Eugene, OR). Indirect Immunofluorescence Analysis.Cells were trans-Zeatin fixed with 3% paraformaldehyde and 0.025% glutaraldehyde, permeabilized with PBS containing 0.1% saponin, and analyzed by indirect immunofluorescence. Cells were first incubated 30 min with primary antibodies, followed by 30 min with TRITC- or FITC-conjugated secondary antibodies (Cappel). Phalloidin (0.5 g/ml) conjugated to either TRITC (Sigma) or FITC (Sigma) was used to label F actin. Cells were viewed with a confocal laser scanning microscope ((1996) . Cells were grown for 2 d on Formvar-coated gold grids, washed with minimum essential medium and 20 mM HEPES, and allowed to internalize for 2 h in type II HRP (Sigma) at a final concentration of 7 mg/ml. Cells were rapidly cooled at 0C and washed with minimum essential medium and 20 mM HEPES. The endocytic compartments containing internalized HRP were cross-linked by incubation for 30 min at 0C in 1.5 mg/ml DAB, 70 mM NaCl, 50 mM ascorbic acid, 20 mM HEPES, and 0.02% H2O2. After removing the excess of DAB by extensive washing with 80 mM piperazine-for 10 min, and the crude membrane fraction contained in the postnuclear supernatant was resuspended in 1.18 M sucrose loaded under a layer of 1 1 M sucrose and a layer of 0.25 M sucrose according to the method of Gorvel (1991) . The gradient was centrifuged 1 h at 130,000 (1987) and centrifuged 20 min at 22,500.
