Ao. Johnson et al., A di-leucine sequence and a cluster of acidic amino acids are required fordynamic, retention in the endosomal recycling compartment of fibroblasts, MOL BIOL CE, 12(2), 2001, pp. 367-381
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.