Furin, a subtilisin-like eukaryotic endoprotease, is responsible for proteolytic cleavage of cellular and viral proteins transported via the constitutive secretory pathway. Cleavage occurs at the C-terminus of basic amino acid sequences, such as R-X-K/R-R and R-X-X-R. Furin was found predominantly in the trans-Golgi network (TGN), but also in clathrin-coated vesicles dispatched from the TGN, on the plasma membrane as an integral membrane protein and in the medium as an anchorless enzyme. When furin was vectorially expressed in normal rat kidney (NRK) cells it accumulated in the TGN similarly to the endogenous glycoprotein TGN38, often used as a TGN marker protein. The signals determining TGN targeting of furin were investigated by mutational analysis of the cytoplasmic tail of furin and by using the hemagglutinin (HA) of fowl plague virus, a protein with cell surface destination, as a reporter molecule, in which membrane anchor and cytoplasmic tail were replaced by the respective domains of furin. The membrane-spanning domain of furin grafted to HA does not localize the chimeric molecule to the TGN, whereas the cytoplasmic domain does. Results obtained on furin mutants with substitutions and deletions of amino acids in the cytoplasmic tail indicate that wild-type furin is concentrated in the TGN by a mechanism involving two independent targeting signals, which consist of the acidic peptide CPSDSEEDEG783 and the tetrapeptide YKGL765. The acidic signal in the cytoplasmic domain of a HA-furin chimera is necessary and sufficient to localize the reporter molecule to the TGN, whereas YKGL is a determinant for targeting to the endosomes. The data support the concept that the acidic signal, which is the dominant one, retains furin in the TGN, whereas the YKGL motif acts as a retrieval signal for furin that has escaped to the cell surface.

Abstract. We have cloned a bovine eDNA encoding the trans-Golgi network (TGN) protease furin and expressed it via recombinant vaccinia viruses to investigate intracellular maturation. Pulse-chase labeling reveals that the 104-kD pro-furin beating high mannose N-glycans is rapidly processed into the 98-kD protease whose N-glycans remain sensitive to endoglycosidase H for a certain period of time. Furthermore, in the presence of brefeldin A, pro-furin cleavage occurs. From these data we conclude that the ER is the compartment of propeptide removal.Studies employing the ionophore A23187 and DTT show that autocatalysis is Ca 2+ dependent and that it does not occur under reducing conditions. Pro-furin produced under these conditions never gains endo H resistance indicating that it is retained in the ER. Coexpression of furin with the fowl plague virus hemagglutinin in the presence of brefeldin A and monensin reveals that furin has to enter the Golgi region to gain substrate cleaving activity.N-glycans of furin are sialylated proving its transit through the trans-Golgi network. A truncated form of furin is found in supernatants of cells. Truncation is inhibited in the absence of Ca 2+ ions and in the presence of acidotropic agents indicating that it takes place in an acidic compartment of cells.Comparative analysis with furin expressed from cDNA reveals that the truncated form prevails in preparations of biologically active, endogenous furin obtained from MDBK cells. This observation supports the concept that secretion of truncated furin is a physiological event that may have important implications for the processing of extracellular substrates.

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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...