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. 1997 Jun 16;137(6):1255-64.
doi: 10.1083/jcb.137.6.1255.

Structural requirements for basolateral sorting of the human transferrin receptor in the biosynthetic and endocytic pathways of Madin-Darby canine kidney cells

G Odorizzi  1 , I S Trowbridge
Affiliations

Structural requirements for basolateral sorting of the human transferrin receptor in the biosynthetic and endocytic pathways of Madin-Darby canine kidney cells

G Odorizzi et al. J Cell Biol. .

Abstract

In polarized Madin-Darby canine kidney (MDCK) cells, the transferrin receptor (TR) is selectively delivered to the basolateral surface, where it internalizes transferrin via clathrin-coated pits and recycles back to the basolateral border. Mutant tailless receptors are sorted randomly in both the biosynthetic and endocytic pathways, indicating that the basolateral sorting of TR is dependent upon a signal located within the 61-amino acid cytoplasmic domain. To identify the basolateral sorting signal of TR, we have analyzed a series of mutant human TR expressed in MDCK cells. We find that residues 19-41 are sufficient for basolateral sorting from both the biosynthetic and endocytic pathways and that this is the only region of the TR cytoplasmic tail containing basolateral sorting information. The basolateral sorting signal is distinct from the YTRF internalization signal contained within this region and is not tyrosine based. Detailed functional analyses of the mutant TR indicate that residues 29-35 are the most important for basolateral sorting from the biosynthetic pathway. The structural requirements for basolateral sorting of internalized receptors from the endocytic pathway are not identical. The most striking difference is that alteration of G31DNS34 to YTRF impairs basolateral sorting of newly synthesized receptors from the biosynthetic pathway but not internalized receptors from the endocytic pathway. Also, mutations have been identified that selectively impair basolateral sorting of internalized TRs from the endocytic pathway without affecting basolateral sorting of newly synthesized receptors. These results imply that there are subtle differences in the recognition of the TR basolateral sorting signal by separate sorting machinery located within the biosynthetic and endocytic pathways.

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Figures

Figure 1
Figure 1
Residues 19–41 of the TR cytoplasmic tail are necessary and sufficient to target TR to the basolateral surface of MDCK cells. Filter-grown MDCK cells expressing wild-type and mutant TR were incubated either apically or basolaterally for 1 h at 4°C with 125I-labeled Tf (4 μg/ml). After washing away unbound Tf, the amount of 125I-labeled Tf specifically bound on each surface was determined (mean ± standard error of three independent experiments).
Figure 2
Figure 2
Residues 19–41 of the TR cytoplasmic tail are necessary and sufficient for basolateral sorting in both the biosynthetic and endocytic pathways. (A) Newly synthesized wild-type and mutant TR expressed in filter-grown MDCK cells were pulse- labeled with 35S-labeled methionine/cysteine for 30 min and then chased to the cell surface for 20 min at 37°C. Receptors at the apical or basolateral surface were then cleaved at 4°C for 30 min with trypsin (100 μg/ml). Trypsin inhibitor (100 μg/ml) was included in the opposite media. B3/25 mAb immunoprecipitates of the ∼70-kD TR extracellular fragment in the media collected from each surface were analyzed on SDS–polyacrylamide gels and quantitated by phosphorimage analysis (mean ± standard error of three independent experiments). No material could be detected in immunoprecipitates from the media containing trypsin inhibitor. (B) Newly synthesized receptors were pulse-labeled as described above and chased at 37°C for 30 min (black bars), 60 min (gray bars), or 120 min (open bars). Trypsin was added to the apical or basolateral surface of the monolayers at 4°C as described above or was included in the apical or basolateral media during the 37°C chase. TR external domain fragments were immunoprecipitated and analyzed by SDS-PAGE as described above. (C) Filter-grown MDCK cells expressing wild-type and mutant TR were incubated either apically or basolaterally at 37°C for 1 h with 125I-labeled Tf. Monolayers were then washed at 4°C, and surface-bound 125I-labeled Tf was removed with deferoxamine mesylate. Cells were then incubated at 37°C for 90 min, and the appearance of 125I-labeled Tf in the apical and basolateral media was determined (mean ± standard error of three independent experiments). More than 90% of the internalized 125I-labeled Tf recycled after 90 min at 37°C.
Figure 3
Figure 3
Residues 29–41 of the TR cytoplasmic tail are the most important for basolateral sorting of newly synthesized TR. Newly synthesized wild-type and mutant TR expressed in filter-grown MDCK cells were pulse-labeled and chased to the cell surface, and the ∼70-kD TR extracellular fragment derived from receptors at either the apical or basolateral surface was immunoprecipitated and quantitated as described in the legend to Fig. 2 A.
Figure 4
Figure 4
The structural requirements for basolateral sorting of TR in the endocytic pathway differ from the structural requirements for sorting in the biosynthetic pathway. The endocytic pathways of MDCK cells expressing wild-type or mutant TR were loaded with 125I-labeled Tf for 1 h at 37°C from either the apical (A) or basolateral (B) border. Monolayers were then washed at 4°C, and the amount of radiolabel recycled to the apical or basolateral surface after 90 min at 37°C was determined as described in the legend to Fig. 2 C.
Figure 5
Figure 5
TR is sorted efficiently to the basolateral surface upon transplantation of residues 29–41 to a more membrane-distal position in the cytoplasmic tail. (A) The amino acid sequence of the TR cytoplasmic tail depicting the transplantation of residues 29– 41 to a more membrane-distal position located between Pro-17 and Leu-18. (B) The fraction of newly synthesized and internalized mutant 29–41 transplant TR sorted basolaterally was determined as described in the legend to Fig. 2.
Figure 6
Figure 6
Features of the TR basolateral sorting signal. Residues 19–41, which are necessary and sufficient for basolateral sorting of TR, are shown. Each of the two four-residue sequences within this region that are predicted to adopt a tight turn conformation, the YTRF internalization signal and the sequence GDNS (Collawn et al., 1990), are underlined. The region most important for basolateral sorting is within residues 29–41, and the TR basolateral sorting signal is neither tyrosine based nor colinear with the YTRF internalization signal. Also indicated is the four-residue sequence RQVD, which is similar to the sequence RNVD found to be important for basolateral sorting of pIgR (Aroeti et al., 1993). However, the sequence RQVD is not required for basolateral sorting of TR, and neither Val-29 nor Val-36 is important for activity of the TR basolateral sorting signal.
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