Two independent targeting signals in the cytoplasmic domain determine trans-Golgi network localization and endosomal trafficking of the proprotein convertase furin.

Abstract

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.

The PC6B Cytoplasmic Domain Contains Two Acidic Clusters That Direct Sorting to Distincttrans-Golgi Network/Endosomal Compartments

“…In the case of furin and the LDL receptor, the acidic motifs alone are not sufficient to support normal localization (Matter et al, 1994;Schäfer et al, 1995). Indeed, a CCP recruitment motif is also required to form a bipartite localization signal and allow efficient internalization from the cell surface (Matter et al, 1994;Jones et al, 1995;Schäfer et al, 1995). Our results with the IS mutations indicate that the localization of PC6B also requires an intact internalization motif.…”

“…To examine the sorting potential of the two ACs, they were analyzed independently. In the case of furin and the LDL receptor, the acidic motifs alone are not sufficient to support normal localization (Matter et al, 1994;Schäfer et al, 1995). Indeed, a CCP recruitment motif is also required to form a bipartite localization signal and allow efficient internalization from the cell surface (Matter et al, 1994;Jones et al, 1995;Schäfer et al, 1995).…”

“…The dynamic localization of furin within the TGN/endosomal system allows the protease to activate a large number of proproteins in multiple compartments. The cytoplasmic domain (cd) of furin is both necessary and sufficient to direct the TGN localization and recycling of the enzyme (Bosshart et al, 1994;Chapman and Munro, 1994;Molloy et al, 1994;Jones et al, 1995;Schäfer et al, 1995;Voorhees et al, 1995). A single cluster of acidic residues, which contains a pair of serines phosphorylated by casein kinase 2 (CK2) in vivo, is a key determinant for directing furin localization (Jones et al, 1995;Takahashi et al, 1995;Dittié et al, 1997;Molloy et al, 1998;Wan et al, 1998).…”

“…These potential targeting signals include two tyrosine-based and one di-leucine-based clathrin-coated pit (CCP) recruitment motifs, as well as clusters of acidic residues. Similar acidic clusters (ACs) have been shown to direct several distinct sorting steps in the TGN/endocytic pathway, including localization to the TGN (furin, carboxypeptidase D, and varicellar zoster virus glycoprotein E; Schäfer et al, 1995;Voorhees et al, 1995;Alconada et al, 1996;Zhu et al, 1996;Eng et al, 1999), basolateral sorting (low-density lipoprotein [LDL] receptor and furin; Matter et al, 1994;Simmen et al, 1999), and delivery of lysosomal hydrolases (both the cation-dependent and cation-independent [CI] mannose-6-phosphate receptors [MPRs]; Chen et al, 1993;Mauxion et al, 1996;Boker et al, 1997;Schweizer et al, 1997).…”

Structures of vesicular stomatitis virus glycoprotein: membrane fusion revisited

“…Second, the folding back of the C-terminal region onto a trimeric N-terminal region leads to the formation of a postfusion protein structure with the outer regions zipped up against the inner trimeric core [2]. For both class I and class II fusion proteins that trigger membrane merger at low pH, the proteolytic cleavage priming the proteins to undergo their low-pH-induced conformational change occurs in the trans-Golgi network or at the host cell surface [44,51,52]. This precludes premature activation of the fusion protein in the acidic compartments of the Golgi apparatus.…”