Sorting of Furin at the Trans-Golgi Network

Abstract

The eukaryotic subtilisin-like endoprotease furin is found predominantly in the trans-Golgi network (TGN) and cycles between this compartment, the cell surface, and the endosomes. There is experimental evidence for endocytosis from the plasma membrane and transport from endosomes to the TGN, but direct exit from the TGN to endosomes via clathrin-coated vesicles has only been discussed but not directly shown so far. Here we present data showing that expression of furin promotes the first step of clathrin-coat assembly at the TGN, the recruitment of the Golgi-specific assembly protein AP-1 on Golgi membranes. Further, we report that furin indeed is present in isolated clathrin-coated vesicles. Packaging into clathrin-coated vesicles requires signal components in the furin cytoplasmic domain which can be recognized by AP-1 assembly proteins. We found that besides depending on the phosphorylation state of a casein kinase II site, interaction of the furin tail with AP-1 and its 1subunit is mediated by a tyrosine motif and to less extent by a leucine-isoleucine signal, whereas a monophenylalanine motif is only involved in binding to the intact AP-1 complex. This study implies that high affinity interaction of AP-1 or 1 with the cytoplasmic tail of furin needs a complex interplay of signal components rather than one distinct signal. The trans-Golgi network (TGN)1 constitutes the sorting station where soluble and membrane proteins, targeted to different post-Golgi compartments, are packed into distinct carrier vesicles. Three TGN exit routes are known: constitutive transport to the cell surface, transport to secretory granules in cells with a regulated secretory pathway, and transport to endosomes (for review, see Ref. 1). The last pathway is followed primarily by lysosomal enzymes bound to the mannose 6-phosphate receptors (MPRs) by their mannose 6-phosphate residues (for review, see Ref.2). Vesicles leaving the TGN for subsequent transport to the endosomes are clathrin-coated. Formation of these clathrin-coated vesicles (CCVs) involves binding of AP-1 Golgi-specific assembly proteins to the TGN membrane. AP-1 binding is regulated by the ADP-ribosylation factor ARF-1, a small GTP-binding protein (3, 4), and requires the presence of transmembrane proteins, among which MPRs are the major constituents (5-7). CCVs are transport intermediates of vesicular traffic not only from the TGN to endosomes, but also from the plasma membrane to endosomes. Golgi-and plasma membrane-derived CCVs can be distinguished by their different sets of assembly proteins; AP-1 complexes are restricted to coated buds and vesicles of the TGN, whereas AP-2 complexes act in endocytosis at the plasma membrane. Both adaptor complexes are heterotetrameric. The AP-1 complex is composed of two 100-kDa subunits, ␥ and ␤1 adaptin, a medium subunit 1 (47 kDa), and a small subunit 1 (19 kDa). The AP-2 complex is of similar size and composition with two large subunits, ␣ and ␤2 adaptin (100 kDa), in association with a 2 (50 kDa) and a 2 subunit (17 kDa) (for r...

Recycling of Furin from the Plasma Membrane

“…For furin tail sorting signals YKGL 765 , LI 760 , and Phe 790 , it has been shown that they are essential for internalization of furin from the plasma membrane (5-7, 19, 20) and that they are also recognized by Golgi-specific AP-1 assembly proteins for subsequent sorting into clathrin-coated vesicles at the TGN (21). Phosphorylation of both serine residues within the acidic TGN localization signal CPSDSEEDEG 783 by CKII enhances recruitment of AP-1 on Golgi membranes in vivo and is important for high affinity AP-1 and 1 binding to the furin tail in vitro (21,30).…”

“…Efficient recycling depends on sequence motifs in its cytoplasmic domain (4,5,35,36). A tyrosine-based motif, a leucine-isoleucine motif, and a monophenylalanine signal mediate internalization at the plasma membrane (5-7, 19, 20) and also interaction with AP-1 Golgi-specific assembly proteins and thus sorting into clathrin-coated vesicles at the TGN (21). On the other hand, an acidic cluster is required for retrieval from endosomes to the TGN (5)(6)(7)19).…”

“…In Vitro Binding Assay-The in vitro binding assay was performed as described (21). Briefly, the 2-chain was in vitro translated in the presence of 35 S-labeled methionine (Amersham Pharmacia Biotech) using the pBluescript-2 DNA as template and a coupled in vitro transcription translation kit (Promega).…”

“…Cells were fixed with 4% paraformaldehyde in PBS and quenched with 50 mM NH 4 Cl in PBS. For co-localization of furin and TGN38, NRK cells were fixed with acetone/ methanol and then incubated with a furin-specific antiserum from rabbit directed against the cytoplasmic tail (diluted 1:500 in PBS) (21) and anti-TGN38 monoclonal antibody (diluted 1:100) (Transduction Laboratories). The primary antibodies were detected by incubation with FITC-conjugated anti-rabbit IgG from swine (Dako) and rhodamineconjugated anti-mouse antibody from goat (Jackson Dianova), both diluted 1:100 in PBS.…”

“…17 and 18). The cytosolic domain of furin contains a tyrosine-based (YKGL 765 ) sorting signal, a leucine-based (LI 760 ) motif, and a monophenylalanine (Phe 790 ) motif, which have been shown to be essential for the internalization of this protein from the cell surface (5-7, 19, 20) and also for interaction with AP-1 Golgi-specific assembly proteins and thus sorting into clathrin-coated vesicles at the TGN (21). The involvement in different sorting processes is an important characteristic of such targeting signals.…”