Addition of excess purified Tlg2TMDp blocked inhibition by anti-Tlg2p serum (Fig. of Ste13HA processing. Fourfold scaled up reactions were incubated under reaction conditions. At the indicated occasions, duplicate 10-l volumes were removed, and the amount of Ste13HA processed was decided as described in Materials and methods. To directly establish that membrane fusion had occurred and Kex2p and Ste13HA were cointegrated into the same membranes at the end of the reaction, product membranes were immunoisolated using TIMP3 affinity purified antibodies Rifampin directed against the Kex2p cytosolic tail (Redding et al., 1991) by a method that results in quantitative isolation of membranes made up of Kex2p (Sipos and Fuller, 2001). If processing of Ste13HA by Kex2p resulted from the fusion of compartments made up of the two enzymes, then isolation of Kex2p-containing compartments should result in coisolation of DPAP activity. MSS membranes from the Kex2p- and Ste13HA-expressing strains were incubated under reaction conditions either together (Fig. 2 , Mixed) or separately (Fig. 2, Separate). Individual samples were combined at the end of the incubation. Incubation of the membranes together resulted in efficient processing of Ste13HA (35%; Fig. 2 A). The products of both reactions were then added to magnetic beads coated with affinity purified anti-Kex2p tail antibodies. After immunoisolation, beads were assayed for Kex2p and DPAP activity. In both samples, 90% of Kex2p activity Rifampin was associated with the beads after immunoisolation (unpublished data). In Kex2p-containing membranes immunoisolated from the separate reaction, no measurable DPAP activity was detected, whereas, 12% of the total DPAP activity from the mixed reaction was associated with the beads (Fig. 2 B). This indicated that this observed Ste13HA processing correlated with membrane fusion, resulting in membranes made up of both Kex2p and DPAP activity. Open in a separate window Physique 2. Ste13HA processing represents a membrane fusion event; DPAP activity is usually associated with immunopurified Kex2p vesicles. (A) Mixed reaction was under standard reaction conditions. Control reaction (Separate) was set up as two half reactions made up of MSS membranes from either Ste13HA- or Kex2p-expressing strains, which were incubated separately at 30C and then combined on ice. Half of each reaction was used to determine the fraction of Ste13HA processed. (B) The remainder of the mixed and individual reactions was bound to anti-Kex2p beads, washed, and assayed for the percent of total DPAP activity, which remained bound to the beads. The somewhat lower yield of immunoisolated DPAP activity (12%) compared with the fraction processed (35%) likely resulted from fragmentation of the membranes during the isolation procedure. Characteristics of the cell-free fusion reaction The cell-free membrane fusion that we observed exhibited features characteristic of other Rifampin cell-free biological membrane fusion reactions. First, although Ste13HA processing in the crude system did not require addition of exogenous ATP (Fig. 3 A), omission of the ATP regeneration system completely blocked processing (Fig. 3 A), indicating that the reaction was ATP dependent. Second, processing of Ste13HA occurred with maximal efficiency between 25 and 30C and was inhibited at temperatures below 15C or above 35C (Fig. 3 B), similar to the temperature dependence of other cell-free biological membrane fusion reactions (Baker et al., 1988; Latterich and Schekman, 1994). Third, dilution of the MSS membranes progressively inhibited Ste13HA processing, indicating that membrane concentration was important for efficient fusion (Fig. 3 C). Fourth, addition of Triton X-100 (1% wt vol?1) completely inhibited Kex2p processing of Ste13HA even though both Kex2p and DPAP were fully active under these conditions (unpublished data), indicating that membrane integrity was essential for efficient processing (Fig. 3 D). Furthermore, this result shows that for processing to be observed in this dilute system Kex2p must be coconcentrated along with the substrate Ste13HA in the lumenal space of fused late Golgi membrane vesicles. Taken together, the characteristics of the reaction suggested that the observed Kex2p-dependent processing of Ste13HA represented a biologically relevant vesicular transport or membrane fusion event between membrane compartments containing Kex2p and Ste13HA. Open in a separate window Figure 3. Characterization of in vitro TGN membrane fusion. (A) Processing of Ste13HA requires an ATP Rifampin regeneration system. Control reactions (Kex2p) included 1.5 mM exogenous Mg-ATP, 40 mM phosphocreatine, and 0.125 mg ml?1 creatine kinase. Omission of Mg-ATP (?ATP), or phosphocreatine/creatine kinase, (?Regen) is indicated. (B) Temperature dependence of Ste13HA processing in vitro. Reactions.