ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 326, No. 1, February 1, pp. 64–72, 1996 Article No. 0047 Characterization of Rab5:Q79L-Stimulated Endosome Fusion 1 M. Alejandro Barbieri,* Guangpu Li,* Luis S. Mayorga,* , † and Philip D. Stahl* ,2 *Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110; and †Instituto de Histologı´a y Embriologı´a–CONICET, Facultad de Ciencias Me ´dicas, Universidad Nacional de Cuyo, 5500 Mendoza, Argentina Received July 3, 1995, and in revised form October 20, 1995 The receptor-mediated internalization and transport of macromolecules from the cell surface into intracellu- Fusion of intracellular membrane-bound compart- lar compartments appear to be mediated by a series of ments is a common step in the transport of macromol- vesicular fusion and budding events (1 – 3). Reconstitu- ecules along the endocytic and secretory pathways. Previous work has shown that GTPgS stimulates en- tion of fusion in several mamalian cell-free systems has dosome fusion in the presence of low concentrations been useful for identifying factors required for endocy- of cytosol. In this study, we have characterized the tosis (4–7). In vitro endosome fusion requires ATP, effect of rab5:Q79L, a mutant with reduced GTPase salt, and cytosolic and membrane-bound proteins, in- activity, on endosome fusion in a cell-free assay. cluding a NEM-sensitive fusion protein (NSF), 3 a phos- rab5:Q79L stimulates in vitro endosome fusion. The pholipase A2 (PLA2) activity, and several GTP-binding stimulatory effects required ATP, were blocked by N- proteins (8, 9). A role for multiple GTP-binding proteins ethylmaleimide (NEM) and anti-NEM-sensitive fusion in endosome fusion was suggested by the observation (NSF) protein antibody, but could proceed in the ab- that GTPgS, a nonhydrolyzable form of GTP, has a sence of cytosol. Stimulation of fusion with rab5:Q79L dual effect on in vitro endosome fusion depending on led to rapid inactivation of the vesicles when tested assay conditions (10 – 12). At very low levels of cytosolic in a second incubation for fusogenic activity. By elec- proteins, normally insufficient to support vesicle dock- tron microscopy, endosomes connected by tubular ing and fusion, GTPgS stimulates in vitro vesicle fu- structures were frequently observed in the presence sion, whereas in the presence of high concentrations of of rab5:Q79L. Rab5:Q79L promoted fusion only among cytosol, GTPgS inhibits in vitro endosome fusion (10, early endosomes; when the ligands were chased into 12). These data point to multiple GTPases. Indeed, more mature endocytic compartments, fusion was work from several laboratories has shown that both not observed. Phospholipase A2 inhibitors blocked heterotrimeric (G proteins) and monomeric GTP-bind- rab5:Q79L-stimulated fusion. The results indicate that ing proteins of the ARF and rab families are involved in rab5:Q79L promotes fusion by activating factors al- intracellular membrane trafficking during endocytosis ready present in the membranes and that NSF and (13–17). phospholipase A2 activities are required downstream The rab family of GTPases plays a central role in of rab5.  1996 Academic Press, Inc. intracellular transport (16 – 20). These proteins are Key Words: intracellular transport; membrane fu- specifically localized in intracellular compartments in- sion; endocytosis; GTPases. cluding the endoplasmic reticulum (rab1 and rab2), cis- Golgi (rab9), early endosomes (rab4 and rab5), and late 1 This work was supported by NIH Grants AI 20015 and GM 42259. 3 Abbreviations used: NEM, N-ethylmaleimide; NSF, NEM-sensi- tive fusion protein; PLA2, phospholipase A2; GDI, GDP dissociation L.S.M. holds a Rockefeller Foundation Biotechnology Fellowship and G.L. is supported by a fellowship from the Parker B. Francis Founda- inhibitor; REP-1, rab escort protein; DNP-b-glucuronidase, dinitro- phenol-derivatized b-glucuronidase; anti-DNP IgG, anti-dinitrophe- tion. 2 To whom correspondence should be addressed at Washington nol IgG; GGTase, geranylgeranyl transferase; DTT, dithiothreitol; GGPP, geranylgeranyl pyrophosphate; PMSF, phenylmethylsulfonyl University School of Medicine, Department of Cell Biology and Physi- ology, Box 8228, 660 S. Euclid Avenue, St. Louis, MO 63110. Fax: fluoride; BSA, bovine serum albumin; ECL, enhanced chemilumines- cence; BEL, bromoenol lactone; DMSO, dimethyl sulfoxide. (314) 362-7463. 64 0003-9861/96 $12.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved.