(A, B, C) Blocking early steps of sphingolipid synthesis exacerbates GARP-associated growth defects. (A) GARP mutants are sensitive to IPC synthase inhibition. Wild-type, vps51Δ, vps52Δ, vps53Δ, and vps54Δ cells were spotted on control plates and plates containing 0.05 μM aureobasidin A. (B) GARP mutants are sensitive to ceramide synthase inhibition. Wild-type, vps51Δ, vps52Δ, vps53Δ, and vps54Δ cells were spotted on control plates and plates containing 100 μM fumonisin B1. (C) VPS53 mutants are sensitive to overexpression of the alkaline ceramidase Ypc1. Wild-type or vps53Δ cells harboring an empty plasmid or a plasmid encoding YPC1 under control of the GAL10 promoter were spotted on glucose- or galactose-containing plates. (D) GARP mutants are sensitive to high levels of long-chain bases, early sphingolipid intermediates. Wild-type, vps53Δ, and vps54Δ cells were spotted on control plates or plates containing 30 μM phytosphingosine (PHS). (E) GARP complex deficiency results in an accumulation of long-chain bases. The lipidomic analysis of sphingolipids from vps53Δ (black) compared with wild-type strains (white) is shown. LCB = long chain base; CER = ceramide; IPC = inositolphosphorylceramide; MIPC = mannosylinositolphosphorylceramide; M(IP)2C = mannose-(inositol-P)2-ceramide. *p < 0.05; n.s. not significant (F, G) Long-chain bases in GARP mutants are reduced upon myriocin treatment but remain elevated. The levels of (F) dihydrosphingosine (DHS) and (G) PHS from wild-type (white bars), vps52Δ (light gray bars), vps53Δ (dark gray bars), and vps54Δ cells (black bars) to myriocin treatment is plotted as fold change from wild-type. *p < 0.05; n.s. not significant (H) Orm1/2 proteins are hyperphosphorylated in vps53Δ mutants. Orm1-HA expressing wild-type or vps53Δ cells were analyzed by Western blotting against the HA tag or PGK1 as control. Wild-type cells were treated with 5 μM myriocin as indicated. Treatment of the cell lysates with λ-phosphatase as indicated.