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

Abstract

The mammalian proprotein convertases (PCs) are a family of secretory pathway enzymes that catalyze the endoproteolytic maturation of peptide hormones and many bioactive proteins. Two PCs, furin and PC6B, are broadly expressed and share very similar cleavage site specificities, suggesting that they may be functionally redundant. However, germline knockout studies show that they are not. Here we report the distinct subcellular localization of PC6B and identify the sorting information within its cytoplasmic domain (cd). We show that in neuroendocrine cells, PC6B is localized to a paranuclear, brefeldin A-dispersible, BaCl 2 -responsive post-Golgi network (TGN) compartment distinct from furin and TGN38. The 88-amino acid PC6B-cd contains sorting information sufficient to direct reporter proteins to the same compartment as full-length PC6B. Mutational analysis indicates that endocytosis is predominantly directed by a canonical tyrosinebased motif (Tyr 1802 GluLysLeu). Truncation and sufficiency studies reveal that two clusters of acidic amino acids (ACs) within the PC6B-cd contain differential sorting information. The membrane-proximal AC (AC1) directs TGN localization and interacts with the TGN sorting protein PACS-1. The membrane-distal AC (AC2) promotes a localization characteristic of the full-length PC6B-cd. Our results demonstrate that AC motifs can target proteins to distinct TGN/endosomal compartments and indicate that the AC-mediated localization of PC6B and furin contribute to their distinct roles in vivo.

Post-translational Processing of β-Secretase (β-Amyloid-converting Enzyme) and Its Ectodomain Shedding

“…Nterminal sequencing confirmed that this cleavage occurs at the site 42 RLPR 45 2 of pro-BACE sulfated at one or more of its carbohydrate moieties. The observations that sulfation of sugars occurs in the TGN (32) and that PCs, except perhaps PC5-B (34), are active only in this compartment or beyond indicated that processing of pro-BACE to BACE occurs either in the TGN or in post-TGN vesicles. This conclusion is consistent with those of Huse et al (40) who recently reported that BACE F is rapidly and efficiently transported to the Golgi apparatus and to a distal secretory pathway.…”

The Cytoplasmic Domain of Vamp4 and Vamp5 Is Responsible for Their Correct Subcellular Targeting

“…Signals based on di-Leu and/or acidic residues have been shown for other proteins, including sortilin, low density lipoprotein receptor-related protein 3 (LRP3), cation-independent mannose 6-phosphate receptor (CI-M6PR), cation-dependent mannose 6-phosphate receptor (CD-M6PR), ␤-site APP-cleaving enzyme 2 (BACE2), p55 tumor necrosis factor receptor 1 (TNFR1p55), furin, and PC6B (43)(44)(45)(46)(47)(48). In these other proteins, the acidic cluster is usually found upstream of the di-Leu motif, which is usually near the C terminus.…”

Proprotein Convertase Furin Interacts with and Cleaves Pro-ADAMTS4 (Aggrecanase-1) in the trans-Golgi Network

“…All constructs were confirmed by DNA sequencing. The expression vectors for furin were constructed as described previously (11,21,22). ␣1PDX was described previously (32).…”