Amphipathic helix α21 is indispensable for normal Sly1 function. (A) CONSURF analysis of evolutionary conservation within the Sly1 loop. Helix α21 is the most highly conserved portion of the loop. Locations of gain-of-function mutations, and hydrophobic residues within the loop are indicated, as are the five substitutions in the Sly1-pα21 mutant. (B) Position of helix α21 within Sly1. Note that no gain-of-function mutations within α21 have been identified. The loop is purple; the domain 3a templating domain is yellow. (C) Helix α21 and residues immediately upstream have the potential to fold into a strongly amphipathic α-helix. The helical wheel renderings comprise the region underlined in black and were produced using HELIQUEST; hydrophobic moment (µH) is indicated. (D) Growth phenotypes of cells carrying sly1-pα21, SLY1-20-pα21, and other alleles were assayed in a sly1∆ strain with a SLY1 balancer plasmid, which is ejected in the presence of 5-FOA. (E–J) RPL fusion with (E–G) Sly1-pα21 and (H–J) the compound mutant Sly1-20-pα21. For reference, fusion is also plotted for Sly1 and Sly1^∆loop. Reactions were set up with (E and H) 0% PEG, Sec17 and Sec18 (100 nM each), and ATP (1 mM); (F and I) 3% PEG and no Sec17, Sec18 (100 nM each), or ATP; or (G, J, and F) 0% PEG, Sec17 and Sec18 (100 nM each), and ATP (1 mM). Fusion was initiated at time = 0 by adding Sly1 or its mutants, at the concentrations indicated in the legends at the right side of the figure. Points show mean ± SEM from three or more independent experiments; in many cases the error bars are smaller than the symbols. Gray lines show least-squares nonlinear fits of a second-order kinetic model. Lipid mixing traces for panels G and J are presented in Fig. S4.