(A) SGA screen summary showing examples of hits (quadruplicate spots are highlighted) on 5-FOA plates (left panel), screen outcomes from two independent sec31-GS₁₃ screens and overlap with sec13Δ screen hits (middle panel), and a list of overlap hits (right panel) that were further confirmed by direct crosses with sec13Δ or sec13Δ erp1Δ (*) strains. ERP2 is functionally related to other hits (highlighted in red) but was identified only in sec31-GS₁₃ screens. (B) Most confirmed BST genes act in GPI-AP remodeling and trafficking pathways. EMP24 and GUP1, though not identified in the screens, conferred bst phenotypes upon direct testing. (C) Growth phenotypes of bstΔ sec13Δ strains generated by direct crosses on 5-FOA at 25°C. (*) The phenotypes of gup1Δ sec13Δ and per1Δ sec13Δ depend on the strain background. (D) Maturation (trafficking) of the GPI-AP Gas1p in p24 and erv29Δ mutants, as revealed by pulse-chase experiments. Maturation of a soluble protein, CPY, is shown for comparison; ‘p’ marks precursor (ER) forms, “m” indicates mature (post-Golgi) forms.

(A) Structural representation of Sec31p showing the flexible span between the β-propeller and ACE domains, including a region (dotted line) not visualized in the crystal structure. (B) Plasmids encoding Sec31p mutants – psec31-Δblade (Δ379–412) or psec31-Δhinge (Δ332–412) – were introduced into sec31Δ and sec31Δ emp24Δ strains as indicated and grown on 5-FOA at 30°C and 37°C. (C) Sec31p-Δhinge was recruited to COPII-containing liposomes as described in Fig. 1D. (D) Sec13p-sfGFP (left and middle panels) redistributed from ER exit sites in wild-type cells (left panel) to the nuclear envelope and cytoplasm in a sec31-Δhinge mutant (middle panel); Sec24p-sfGFP localized normally in a sec13Δ strain expressing Sec31p-Δhinge (right panel). Scale bar is 2 μm. (E) The sec31-Δhinge mutant complements a sec13Δ strain, permitting growth on 5-FOA at 25°C. (F) Maturation of Gas1p and CPY maturation was assessed as described in Fig. 2D.

(A) Schematic and structural representations of Sec31p (green) and Sec13p (orange) showing the Sec31p DIM (blue), β-propeller (light green) and ACE (dark green) domains. Residues (W386D and A387K) altered in the Sec31p-DK mutant are indicated and numbering denotes Sec31p residues. (B) Schematic of the Sec31-Sec13p fusion, which was introduced into sec31Δ and sec13Δ strains as indicated and grown on media containing 5-FOA to counterselect for the wild-type plasmid-borne copies of SEC13 or SEC31. (C) A sec31Δ strain containing a plasmid-borne sec31-GS₁₃ and sec31-DK mutant as the sole copy of SEC31 was viable only in bst mutant backgrounds. (D) Synthetic liposomes were incubated with the COPII coat proteins and guanine nucleotides indicated and coat assembly assessed by liposome flotation and SDS-PAGE. Numbered lanes contain liposome-bound proteins; * denotes a degradation product of Sec31p. (E) Microsomal membranes (T – 10% of total input membranes) purified from bst1Δ cells were incubated with Sar1p and Sec23/24p, supplemented with Sec13p, Sec31p and guanine nucleotides as indicated; vesicles released were purified and analyzed by immunoblotting against cargo proteins Sec22p and Erv46p.

(A) Reticulons, which maintain curvature of the ER, are not required for the bst phenotype because bst1Δ sec13Δ cells are viable when Yop1p and Rtn1p are deleted. (C) Likewise, long-chain fatty acid synthases Sur4p and Fen1p are not required for generation of curvature during COPII vesicle formation.