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. 2007 Nov 15;26(24):4935–4945. doi: 10.1038/sj.emboj.7601915

Figure 4.

Figure 4

Nyv1p-CCIIM engages in vacuole fusion. (A) The Sec18p/Vam7p-mediated fusion of Nyv1p-CCIIM vacuoles is inhibited by antibodies to Nyv1p, relieved only by recombinant sNyv1p. Vacuoles from BJ3505 NYV1-CCIIM and DKY6281 NYV1-CCIIM were incubated in fusion reactions containing both Sec18p and Vam7p at 27°C in the presence of indicated proteins. After 90 min, reactions were assayed for fusion. Data represent mean±s.e.m. (n=3). To optimize the chance of seeing relief from αNyv1p inhibition, we employed 3.3 μM GST-Vam7p (lanes 8 and 13), a level which itself often causes some fusion inhibition (lane 13). (B) The zero-layer arginine of Nyv1p-CCIIM is important for Nyv1p-CCIIM vacuole fusion. Standard fusion assays (27°C, 90 min) bore BJ3505 nyv1Δ vacuoles and either DKY6281 NYV1-CCIIM or DKY6281 NYV1-CCIIM R192Q vacuoles. Vam7p (638 nM), his6-Sec18p (63.8 nM), and anti-Vam3p (444 nM) were added where indicated.