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Abstract. Septin proteins are necessary for cytokinesis in budding yeast and Drosophila and are thought to be the subunits of the yeast neck filaments. To test whether septins actually form filaments, an immunoaffinity approach was used to isolate a septin complex from Drosophila embryos. The purified complex is comprised of the three previously identified septin polypeptides Pnut, Sep2, and Sepl. Hydrodynamic and sequence data suggest that the complex is composed of a heterotrimer of homodimers. The complex copurifies with one molecule of bound guanine nucleotide per septin polypeptide. It binds and hydrolyzes exogenously added GTP. These observations together with conserved sequence motifs identify the septins as members of the GTPase superfamily. We discuss a model of filament structure and speculate as to how the filaments are organized within cells. CVTOKINESIS involves the concerted activity of cytoskeletal and membrane systems to create two cells from one. Despite differences in morphology and apparent mechanism, yeast cells and animal cells appear to use a similar set of proteins to accomplish this step in cell division. Actin and the recently identified septin proteins are required in both systems (reviewed by Field, 1994, andLongtine et al., 1996). The septins are a homologous family of proteins identified in budding and fission yeast (Haarer and Pringle, 1987;Ford and Pringle, 1991;Kim et al., 1991; Pringle, J., personal communication), Drosophila (Neufeld and Rubin, 1994;Fares et al., 1995), mammals, (Kato, 1990Nottenburg et al., 1990;Kumar et al., 1992;Nakatsuru et al., 1994), and Xenopus (Glotzer, M., and T. Hyman, personal communication). They appear to be involved in cytokinesis or septum formation, perhaps including the regulation of plasma membrane--cortical cytoskeleton interactions and playing a more general role in cell-surface organization. The four original septin family members, encoded by CDC3, CDCIO, CDCll, and CDC12, were first identified in budding yeast on the basis of mutations affecting the cell division cycle (Hartwell, 1971). The phenotypes of all four mutations are indistinguishable, with defects in bud morphogenesis, cytokinesis, and the localization of chitin deposition. In Drosophila, a mutation in the septin gene pea-

Arf and Rab family GTPases regulate membrane traffic in cells, yet little is known about how they are targeted to distinct organelles. To identify sequences in Arf-1 necessary for Golgi targeting, we examined the localization of chimeras between Arf-1 and Arf-6. Here, we identify a 16–amino acid sequence in Arf-1 that specifies Golgi targeting and contains a motif (MXXE) that is important for Arf-1 binding to membrin, an ER-Golgi SNARE protein. The MXXE motif is conserved in all Arfs known to localize to the Golgi and enables Arf-1 to localize to the early Golgi. Arf-1 lacking these 16 aa can still localize to the late Golgi where it displays a more rapid Golgi-cytosol cycle than wild-type Arf-1. These studies suggest that membrin recruits Arf-1 to the early Golgi and reveal distinct kinetic cycles for Arf-1 at early and late Golgi determined by different sets of Arf regulators and effectors.

The ADP-ribosylation factor 6 (ARF6) GTPase has a dual function in cells, regulating membrane traffic and organizing cortical actin. ARF6 activation is required for recycling of the endosomal membrane back to the plasma membrane (PM) and also for ruffling at the PM induced by Rac. Additionally, ARF6 at the PM induces the formation of actin-containing protrusions. To identify sequences in ARF6 that are necessary for these distinct functions, we examined the behavior of a chimeric protein of ARF1 and ARF6. The 1-6 chimera (with the amino half of ARF1 and the carboxyl half of ARF6) localized like ARF6 in HeLa cells and moved between the endosome and PM, but it did not form protrusions, an ARF6 effector function. Two residues in the amino-terminal half of ARF6, Q37 and S38, when substituted into the 1-6 chimera allowed protrusion formation, whereas removal of these residues from ARF6 resulted in an inability to form protrusions. Interestingly, expression of 1-6 in cells selectively inhibited protrusions induced by wild-type ARF6 but had no effect on ARF6-regulated membrane movement or Rac-induced ruffling. Thus, we have uncoupled two functions of ARF6, one involved in membrane trafficking, which is necessary for Rac ruffling, and another involved in protrusion formation.