Fig. 2. Mislocalization of Arn1p to the plasma membrane in a vps1Δ strain. (A and B) Binding of ferrichrome to Arn1p expressed on the plasma membrane. (A) Strain CWY101, in which all four Arn transporters have been deleted, was transformed with the vector pRS316 (vector), the low-copy-number pArn1-HA (pArn1) to reconstitute endogenous levels of Arn1p, or the high-copy-number pOE-Arn1-HA (pOE Arn1). Cells were grown to mid-log phase in iron-poor medium, then membrane transport and uptake systems were inactivated by incubation with NaN₃ and KF at 0°C. The capacity of Arn1p that is expressed on the plasma membrane to bind [⁵⁵Fe]ferrichrome at 0°C was measured. (B) Strain RH144–3D (VPS+) and the congenic strain CWY102 (vps1Δ) were grown to mid-log phase in rich medium and cell surface binding of [⁵⁵Fe]ferrichrome was measured. (C) Expression of Arn1p in both plasma membrane and intracellular vesicle fractions in a vps1 deletion strain. Strain RH144-3D (VPS+) and the congenic strain CWY102 (vps1Δ) were transformed with pArn1-HA and grown to mid-log phase in rich medium. Cells were lysed, membranes were collected and then separated on discontinuous sucrose gradients. Fractions were collected from the top and subjected to SDS–PAGE and western blotting using antibodies directed against Vps10p to detect late-Golgi membranes (Vps10p), Gas1p to detect plasma membrane (Gas1p) and the HA epitope to detect Arn1p-HA (Arn1p). The arrow indicates an endoplasmic reticulum-derived, immature form of Gas1p. Molecular weight standards are indicated in kDa.