Identification of amino-terminal lysine residues as targets of ubiquitination. (A) Preferential ubiquitination of amino-terminal lysines. (B) Identification of lysine 14 of Arn1p as primary target of ubiquitination. (C) Targeting of amino-terminal lysines at all ferrichrome concentrations. YPH499 was transformed with YEp96 and a plasmid containing either wild-type Arn1-HA (pMetArn1-HA, WT) or mutant forms of Arn1-HA (K11R, K14R, K15R, K259R, K267R, K270R, 3NKR, or 3CKR). YPH499 transformed with pRS316 and YEp96 was used as the no-HA control. YPH499 integrated with pRS404 and transformed with pMetArn1-HA was used as the low-Ub control. Transformants were grown in SC without methionine and with 100 μM copper. FC was added at the last hour to the final concentration of 5 μM (A and B) or as indicated (C). Equivalent numbers of cells were harvested from each culture. Cells were lysed and membranes were purified, detergent solubilized, and subjected to immunoprecipitation with anti-HA antibody. Immunoprecipitates were then subjected to Western blotting using anti-HA antibodies to detect Arn1-HA (top) or anti-Ub antibodies to detect ubiquitinated Arn1 (bottom). Equivalent aliquots of the solubilized membranes used for immunoprecipitations in C were subjected to Western blotting with anti-tubulin antibody as a control. (D) Absence of surface mislocalization in Arn1-3NKR. The strain CWY101, from which all ARN transporters have been deleted, was transformed with pMetArn1-HA, pMetArn1-3NKR-HA, or the empty vector pRS316. Transformants were grown in SC medium, cultures were divided, and 0.02 μM FC was added to one aliquot for the final 2 h of growth. Cells were treated with 20 mM NaN3 and KF, harvested, washed, and mixed with [55Fe-FC] at 100 nM at 0°C for 15 min. Cells were again washed and retained [55Fe-FC] was measured. All samples were prepared in triplicate, and binding assays were repeated three times with similar results. Data from a single representative experiment are shown; error bars represent average deviation.