(A) Liposome flotation assays showing activated Ypt1 and Ypt31 recruit purified Sec7f to liposomes. GTP* = GMP-PNP (active GTPase).
(B) Activated Arf1 and Arl1, but not Ypt6, recruit purified Sec7f to liposomes. *, contaminant. Purified Rab GTPases bind to membranes regardless of their nucleotide state via a 7xHis tag, which is not present on purified Arf1 or Arl1.
(C) Localization of an extra copy of GFP-Sec7 in yeast cells harboring the indicated mutations. For temperature-sensitive mutants, images are shown for both permissive (30°C) and restrictive (37°C) temperatures.
(D-E) Overexpression of indicated GTPases via 2-μm plasmids in temperature-sensitive yeast cells carrying (D) GFP-sec7-1 or (E) RFP-sec7-4 mutant alleles.
(F) Schematic diagram of the Sec7 conserved domain structure and Sec7 truncated constructs; stars denote the approximate locations of the specified mutations. See also Figure S1.