A di-leucine sequence and a cluster of acidic amino acids are required fordynamic, retention in the endosomal recycling compartment of fibroblasts

Insulin-regulated aminopeptidase (IRAP), a transmembrane aminopeptidase, is dynamically retained within the endosomal compartment of fibroblasts. The characteristics of this dynamic retention are rapid internalization from th e plasma membrane and slow recycling back to the cell surface. These specia lized trafficking kinetics result in < 15% of IRAP on the cell surface at s teady state, compared with 35% of the transferrin receptor, another transme mbrane protein that traffics between endosomes and the cell surface. Here w e demonstrate that a 29-amino acid region of IRAP's cytoplasmic domain (res idues 56-84) is necessary and sufficient to promote trafficking characteris tic of IRAP. A di-leucine sequence and a cluster of acidic amino acids with in this region are essential elements of the motif that slows IRAP recyclin g. Rapid internalization requires any two of three distinct motifs: M-15,M- 16, DED64-66, and LL76,77. The DED and LL sequences are part of the motif t hat regulates recycling, demonstrating that this motif is bifunctional. In this study we used horseradish peroxidase quenching of fluorescence to demo nstrate that IRAP is dynamically retained within the transferrin receptor-c ontaining general endosomal recycling compartment. Therefore, our data demo nstrate that motifs similar to those that determine targeting among distinc t membrane compartments can also regulate the rate of transport of proteins from endosomal compartments. We propose a model for dynamic retention in w hich IRAP is transported from the general endosomal recycling compartment i n specialized, slowly budding recycling vesicles that are distinct from tho se that mediate rapid recycling back to the surface (e.g., transferrin rece ptor-containing transport vesicles). It is likely that the dynamic retentio n of IRAP is an example of a general mechanism for regulating the distribut ion of proteins between the surface and interior of cells.