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. 2001 Feb;12(2):367-81.
doi: 10.1091/mbc.12.2.367.

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

A O Johnson  1 , M A Lampson, T E McGraw
Affiliations
Free PMC article

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

A O Johnson et al. Mol Biol Cell. 2001 Feb.
Free PMC article

Abstract

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 the plasma membrane and slow recycling back to the cell surface. These specialized trafficking kinetics result in <15% of IRAP on the cell surface at steady state, compared with 35% of the transferrin receptor, another transmembrane protein that traffics between endosomes and the cell surface. Here we demonstrate that a 29-amino acid region of IRAP's cytoplasmic domain (residues 56--84) is necessary and sufficient to promote trafficking characteristic of IRAP. A di-leucine sequence and a cluster of acidic amino acids within this region are essential elements of the motif that slows IRAP recycling. Rapid internalization requires any two of three distinct motifs: M(15,16), DED(64--66), and LL(76,77). The DED and LL sequences are part of the motif that regulates recycling, demonstrating that this motif is bifunctional. In this study we used horseradish peroxidase quenching of fluorescence to demonstrate that IRAP is dynamically retained within the transferrin receptor-containing general endosomal recycling compartment. Therefore, our data demonstrate that motifs similar to those that determine targeting among distinct membrane compartments can also regulate the rate of transport of proteins from endosomal compartments. We propose a model for dynamic retention in which IRAP is transported from the general endosomal recycling compartment in specialized, slowly budding recycling vesicles that are distinct from those that mediate rapid recycling back to the surface (e.g., transferrin receptor-containing transport vesicles). It is likely that the dynamic retention of IRAP is an example of a general mechanism for regulating the distribution of proteins between the surface and interior of cells.

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Figures

Figure 1
Figure 1
Cytoplasmic sequences of the vpTR constructs used in this study. (A) IRAP's cytoplasmic domain amino acid sequence is shown. Wild-type vpTR contains the entire 109-amino–terminal cytoplasmic domain of IRAP fused to the transmembrane and extracellular domains of the human TR (Johnson et al., 1998). The numbers correspond to the position of residues from the amino terminus. The underlined residues have been mutated by alanine substitution and are discussed in the text. (B) Schematics of the deletion and substitution constructs are shown. The white-boxed regions are IRAP sequences and the numbers refer to the amino acid sequences of IRAP contained in the various constructs. The stippled regions are from the TR. All of the constructs contain the transmembrane and extracellular domains of the TR.
Figure 2
Figure 2
Endocytic trafficking kinetics of the amino-terminal deletion vpTR constructs. (A) Percentage of vpTR deletion constructs on the cell surface at steady state. The results shown are the average values ± SEM from at least three independent determinations. (B) The average recycling rate constants from at least four independent determinations ± SEM are presented. (C) The average internalization rate constants from at least four independent determinations ± SEM are shown. The data are from representative clonal lines expressing the various constructs. The values noted by an asterisk are different from the corresponding value for vpTR with P values < 0.001 (heteroscedastic, two-tailed Student's t test). The internalization rate constants of Δ1 and Δ2vpTR differ from the internalization rate constant of vpTR with a P < 0.1, noted by a double asterisk (heteroscedastic, two-tailed Student's t test).
Figure 3
Figure 3
Recycling kinetics of Δ2 vpTR constructs containing point mutations in LL76–77 or the cluster of acidic amino acids. The average recycling rate constants ± SEM from at least three independent determinations are presented. The data are from representative clonal lines expressing the various constructs. The recycling rate constants of TR and vpTR are presented for comparison. The recycling rate constants of the mutants in the Δ2vpTR background are all different from the recycling rate constant of ΔvpTR with P values < 0.001 (heteroscedastic, two-tailed Student's t test).
Figure 4
Figure 4
Internalization kinetics of Δ2 vpTR constructs containing point mutations in LL76–77 or the cluster of acidic amino acids. The internalization rates of the mutant constructs are expressed as percentages of the internalization rate of Δ2 vpTR. The data are the averages ± SEM from at least three independent determinations. The internalization rate of Δ4 vpTR, expressed as a percentage of Δ2 vpTR, is presented as a measure of the nonconcentrative internalization rate. The internalization rates constants for Δ2vpTR-DED64–66, Δ2vpTR-EED67–69, and Δ2vpTR-YES70–72 are different from the internalization rate constant of Δ2vpTR and from the rate for Δ4 vpTR with P values < 0.001 (heteroscedastic, two-tailed Student's t test).
Figure 5
Figure 5
Endocytic trafficking kinetics of the mutations in the cluster of acidic residues in the full-length IRAP cytoplasmic domain. (A) The average recycling rate constants of vpTR constructs containing point mutations in the acidic sequences. The average recycling rate constants from at least three independent determinations ± SEM are presented. The internalization rate constants of vpTR and vpTR LL76,77AA are presented for comparison. The exocytic rate constants for the various mutants of vpTR are all different from the exocytic rate constant of vpTR with a P < 0.001 (heteroscedastic, two-tailed Student's t test). (B) The average internalization rate constants from at least three independent determinations ± SEM are presented. Data are from representative clonal cell lines expressing the indicated constructs.
Figure 6
Figure 6
Internalization rate constant measurements of full-length vpTR containing mutations in both the cluster of acidic resides and LL76–77. The average internalization rate constants from at least three independent determinations ± SEM are presented. Experiments were performed on representative clonal cell lines expressing the indicated constructs. The internalization rate constant of vpTR is presented for comparison.
Figure 7
Figure 7
Internalization rate constant measurements of full-length vpTR containing mutations in MI15,16 or in both MI15,16 and LL76–77. The data are presented as percentages of the internalization rate of vpTR. The data for Δ1 vpTR and Δ2 vpTRLL76,77AA are presented for comparison. Experiments were performed on representative clonal cell lines expressing the indicated constructs. The internalization rate constant of vpTR MI15,16 differs from that of vpTR with a P < 0.1 (heteroscedastic, two-tailed Student's t test). The values noted by the asterisk differ from the internalization rate constant of vpTR with P < 0.001.
Figure 8
Figure 8
Endocytic trafficking kinetics of vp1–84TR and vp55–84TR constructs. (A) The average ± SEM recycling rate constants determined in at least three independent measurements are shown. The internalization rate constants of vpTR and TR are shown for comparison. ▪, measured in the absence of insulin; □, from cells incubated with 500 nM insulin. (B) Percentage of vpTR deletion constructs on the cell surface. The results shown are the average values ± SEM from at least three independent determinations.
Figure 9
Figure 9
Colocalization of NBD-SM and Cy3-Tf. Uptake of NBD-SM (green) and Cy3-Tf (red) is shown in cells expressing the TR (A–C), vpTR (D–F), LL76,77AA mutant (G–I), DED64–66AAA mutant (J–L), LL76,77AA and DED64–66AAA double mutant (M–O), or vp55–84TR (P–R) constructs. Scale bar, 10 μm. Images are single optical sections (1-μm thickness) from the confocal microscope.
Figure 10
Figure 10
Colocalization of the TR or vpTR with F-WGA shown by fluorescence quenching. Cells were labeled with F-WGA, incubated with either HRP-Tf or unlabeled Tf, and processed for DAB cytochemistry to quench the F-WGA fluorescence as described in MATERIALS AND METHODS. (A–D) F-WGA fluorescence with no HRP and no quenching (A and C) or after quenching due to HRP-Tf uptake (B and D) in cells expressing the TR (A and B) or vpTR (C and D). Arrows, recycling compartments labeled with F-WGA. Scale bar, 10 μm. (E) Total fluorescence intensity per field in cells with or without HRP-Tf uptake (mean ± SE, N = 10 fields, >20 cells per field). (F) Fluorescence intensity per field above a threshold in cells with or without HRP-Tf uptake. The threshold was chosen to include the recycling compartments but exclude peripheral fluorescence (explained in MATERIALS AND METHODS).
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