Figure 1.

Peptidoliposomes to assay AP-1 recruitment in vitro. The maleimide derivative of PE MMCC-DHPE was used to couple synthetic peptides via an N-terminal cysteine to a lipid (A). The peptides used correspond to the cytoplasmic domain of Lamp1 (B, Lamp1Y) or the segment of TGN38 that has previously been shown to contain the functional tyrosine motif (Boll et al., 1996). Lamp1A and TGN38A are the control peptides with the critical tyrosine mutated to alanine. After incubation of peptidoliposomes with AP-1 and with or without ARF1, they were floated from the bottom of a sucrose step gradient (C). Four fractions were collected as indicated, with fraction I containing the floated liposomes with bound proteins and fraction IV including the loading zone with unbound proteins.