Basolateral sorting of furin in MDCK cells requires a phenylalanine-isoleucine motif together with an acidic amino acid cluster

Abstract

Furin is a subtilisin-related endoprotease which processes a wide range of bioactive proteins. Furin is concentrated in the trans-Golgi network (TGN), where proteolytic activation of many precursor proteins takes place. A significant fraction of furin, however, cycles among the TGN, the plasma membrane, and endosomes, indicating that the accumulation in the TGN reflects a dynamic localization process. The cytosolic domain of furin is necessary and sufficient for TGN localization, and two signals are responsible for retrieval of furin to the TGN. A tyrosine-based (YKGL) motif mediates internalization of furin from the cell surface into endosomes. An acidic cluster that is part of two casein kinase II phosphorylation sites (SDSEEDE) is then responsible for retrieval of furin from endosomes to the TGN. In addition, the acidic EEDE sequence also mediates endocytic activity. Here, we analyzed the sorting of furin in polarized epithelial cells. We show that furin is delivered to the basolateral surface of MDCK cells, from where a significant fraction of the protein can return to the TGN. A phenylalanine-isoleucine motif together with the acidic EEDE cluster is required for basolateral sorting and constitutes a novel signal regulating intracellular traffic of furin.

FIG. 1

Furin is sorted to the basolateral cell surface in MDCK cells. MDCK cells expressing human furin were grown on coverslips, fixed, and stained with an antifurin antibody (a) or with an anti-TGN38 antibody (b). Furin is enriched in a perinuclear compartment that resembles the compartment carrying TGN38, and superposition of the two panels indicates extensive colocalization (c, yellow color). Cells not expressing furin do not stain with antifurin (panels a to c, insets). For panels d, e, and f, cells were incubated with antifurin antibody present in the medium for 60 min at 37°C prior to fixation, permeabilization, and staining with a labeled secondary antibody (d) or with anti-TGN38 (e). Merging of the two images reveals little colocalization of internalized antifurin antibodies and endogenous TGN38 (f), probably reflecting lysosomal delivery of cross-linked furin. Untransfected cells do not internalize the antifurin antibodies (panels d, e, and f, insets), indicating that antibody uptake did not occur by fluid-phase endocytosis. For panels g and h, polarized MDCK cell monolayers were incubated at 37°C in the presence of antifurin antibodies added to the apical (g) or basolateral (h) compartment. Antibodies are selectively internalized from the basolateral surface. Bars: 10 μm.

FIG. 2

Amino acid sequences of the cytosolic domains of wild-type furin tail and mutant Tac-furin tail chimeras and their polarized distribution. Amino acid sequences are shown in the single-letter code, and known sorting signals in the furin tail are underlined. Residues are numbered from left to right, with position 1 corresponding to the presumed initiation methionine. The nomenclature of the mutants is according to that of Voorhees et al. (40); TFT is a chimera combining the luminal and cytosolic domains of Tac and the transmembrane domain of furin; TTF and Tac F are chimeras combining the cytosolic tail of furin and the ecto- and transmembrane domains or complete Tac, respectively. Alanine substitutions in the wild-type sequence are shown in boldface. The polarized distribution of the different constructs is summarized to the right, and the number(s) of the figure(s) showing the data for the corresponding construct is given. Where no data is shown in the text, it is indicated whether the distribution was determined by immunofluorescence (IF) and/or radioactive antibody binding (bdg).

FIG. 3

Localization and endocytosis of the Tac-furin tail chimera TTF. MDCK cells expressing TTF grown on coverslips were fixed and stained with an anti-Tac antibody (a) or an anti-TGN38 antibody (b). TTF is enriched in a perinuclear compartment that resembles the compartment carrying TGN38, and superposition of the two panels indicates extensive colocalization (c, yellow color). Cells not expressing TTF do not stain with anti-Tac (panels a, b, and c, arrows). For panels d and e, cells were incubated for 60 min at 37°C in the presence of anti-Tac antibodies to allow for the internalization of antibody and then fixed, permeabilized, and stained with a labeled secondary antibody (d) or with anti-TGN38 (e). Merging the two images reveals significant colocalization of internalized anti-Tac antibodies and endogenous TGN38 (f). Untransfected cells do not internalize anti-Tac (panels d, e, and f, arrows in insets), indicating that anti-Tac uptake did not occur by fluid-phase endocytosis. For panels g, h, and i, polarized MDCK cell monolayers were incubated at 37°C in the presence of anti-Tac antibodies added to the basolateral compartment. Fixed cells were then incubated with a labeled secondary antibody to detect the anti-Tac antibody (g) or stained with an anti-TGN38 antibody (h). Superposition of the two panels indicates extensive colocalization (i, yellow color), indicating that a significant fraction of anti-Tac antibodies internalized from the basolateral surface was retrieved to the TGN. Bars, 10 μm.

FIG. 4

The cytosolic domain of furin is necessary and sufficient for basolateral sorting and requires a signal in the membrane distal half of the tail. MDCK cells expressing TTF (a and b), TTFΔ746 (c and d), Tac F766-793 (e and f), or TTFΔ765 (g and h) were grown as polarized cell monolayers and incubated at 37°C for 30 min with anti-Tac antibody added to the upper (panels a, c, e, and g) or lower (panels b, d, f, and h) compartment. Cells were then washed on ice, fixed, and permeabilized, and anti-Tac antibodies were visualized by using a labeled second antibody. The chimera containing the wild-type tail (TTF) is transported to the basolateral surface (panels a and b), and the deletion of the tail in TTFΔ746 abolishes basolateral delivery (panels c and d). A chimera containing the membrane distal part of the furin tail (Tac F766-793) is expressed on the basolateral surface (panels e and f), whereas TTF Δ765, encoding the membrane proximal half of the tail, is transported apically (panels g and h). Bar, 10 μm.

FIG. 5

Basolateral sorting requires a membrane-distal phenylalanine-isoleucine motif. MDCK cells expressing Tac F766-787 (a and b), Tac F766-785 (c and d), Tac F780-793 (e and f), or Tac F780-787 (g and h) were grown as polarized cell monolayers and incubated at 37°C for 30 min with anti-Tac antibody added to the upper (panels a, c, e, and g) or lower (panels b, d, f, and h) compartment. Cells were then washed on ice, fixed, and permeabilized, and anti-Tac antibodies were visualized by using a labeled second antibody. Tac F766-787, a truncation mutant that carries the FI, is sorted basolaterally (panels a and b), but removal of the FI in Tac F766-785 abolishes basolateral transport (panels c and d). N-terminal truncation mutants containing the FI, Tac F780-793 or Tac F780-787, are delivered basolaterally (panels e through h). Bars, 10 μm.

FIG. 6

The acidic amino acid cluster EEDE and the FI motif but not serine phosphorylation are required for basolateral sorting. MDCK cells expressing Tac F766-793 FI→AA (a and b), Tac F766-793 EEDE→AAAA (c and d), TTF SS→AA (e and f) or TTF SS→DD (g and h) were grown as polarized cell monolayers and incubated at 37°C for 30 min with anti-Tac antibody added to the upper (panels a, c, e, and g) or lower (panels b, d, f, and h) compartment. Cells were then washed on ice, fixed, and permeabilized, and anti-Tac antibodies were visualized by using a labeled second antibody. Mutation of the FI and EEDE but not that of the serines subject to phosphorylation by CKII affects basolateral transport. Bar, 10 μm.

FIG. 7

The EEDE cluster and the FI motif are required for basolateral sorting in the context of the complete furin tail. MDCK cells expressing TTF FI→AA (a and b), TTF Y758A (c and d), TTF EEDE→AAAA (e and f), or TTF Y758A, EEDE→AAAA (g and h) were grown as polarized cell monolayers and incubated at 37°C for 30 min with anti-Tac antibody added to the upper (panels a, c, e, and g) or lower (panels b, d, f, and h) compartment. Cells were then washed on ice, fixed, and permeabilized, and anti-Tac antibodies were visualized by using a labeled second antibody. Mutations affecting either the FI motif or the EEDE motif in TTF FI→AA, TTF EEDE→AAAA, or TTF Y758A, EEDE→AAAA lead to impaired basolateral sorting (panels a, b, and e through h); inactivating the tyrosine-based endocytosis signal in TTF Y758A has no effect on basolateral transport (panels c and d). Bar, 10 μm.

FIG. 8

Quantitation of the steady-state distribution of different Tac-furin tail constructs. MDCK cells expressing the Tac-furin tail chimeras indicated were grown on Transwell filters. To quantitate the surface expression of the different chimeras, radiolabeled anti-Tac antibody was allowed to bind from either the apical or the basolateral compartment on ice. Similar distributions were obtained for different clones expressing different levels of chimeric proteins. Quantitation of the binding for each clone was obtained from three to five independent experiments, each carried out in duplicate or triplicate. Statistical analysis showed that the distribution of constructs containing the FI and EEDE motifs was significantly different from that of Tac, while the distribution of constructs lacking the FI and EEDE significantly differed (P < 0.005, Student’s t test) from that of TTF, confirming the critical role of the FI and EEDE in basolateral sorting.