Memapsin 2 (β‐secretase) cytosolic domain binds to the VHS domains of GGA1 and GGA2: implications on the endocytosis mechanism of memapsin 2

Abstract

Memapsin 2, or L L-secretase, is a membrane-anchored aspartic protease that initiates the cleavage of L L-amyloid precursor protein (APP) leading to the production of L L-amyloid peptide in the brain and the onset of Alzheimer's disease. Memapsin 2 and APP are both endocytosed into endosomes for cleavage. Here we show that the cytosolic domain of memapsin 2, but not that of memapsin 1, binds the VHS domains of GGA1 and GGA2. Gel-immobilized VHS domains of GGA1 and GGA2 also bound to full-length memapsin 2 from cell mammalian lysates. Mutagenesis studies established that Asp 496 , Leu 499 and Leu 500 were essential for the binding. The spacing of these three residues in memapsin 2 is identical to those in the cytosolic domains of mannose-6-phosphate receptors, sortilin and low density lipoprotein receptor-related protein 3. These observations suggest that the endocytosis and intracellular transport of memapsin 2, mediated by its cytosolic domain, may involve the binding of GGA1 and GGA2. ß

Internalization of Exogenously Added Memapsin 2 (β-Secretase) Ectodomain by Cells Is Mediated by Amyloid Precursor Protein

“…The endocytosis of memapsin 2 is thought to require its cytosolic domain and a dileucine motif contained therein (11,26). It was demonstrated recently that the dileucine motif is part of the acid cluster-dileucine (ACDL) sequence DDISLL that binds to the VHS domain of GGA (Golgi-localized ␥-ear-containing ADP-ribosylation factor binding) proteins (17,18). Because GGA proteins also bind clathrin, this interaction appears to mediate the packaging of memapsin 2 to the coated pit for vesicular transport.…”

“…Both proteins are endocytosed into the endosomes, the major site for the hydrolysis of APP by memapsin 2 (9, 11), leading to the production of A␤. The intracellular domain of memapsin 2 contains a signal motif that binds three GGA proteins (17,18). This interaction apparently serves to package memapsin 2 into clathrin-coated vesicles for transportation in the endocytic or/and recycling pathways (17,18).…”

“…The intracellular domain of memapsin 2 contains a signal motif that binds three GGA proteins (17,18). This interaction apparently serves to package memapsin 2 into clathrin-coated vesicles for transportation in the endocytic or/and recycling pathways (17,18). Being located in the lipid raft at the plasma membrane (19,20), memapsin 2 endocytosis is influenced by other components having direct or indirect contact with the protease.…”

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.…”

Direct binding of the Kex2p cytosolic tail to the VHS domain of yeast Gga2p facilitates TGN to prevacuolar compartment transport and is regulated by phosphorylation

“…Biochemical and structural studies of binding between the mammalian GGA-VHS domains and the C-tails of TGN receptors sortilin and two mannose-6-phosphate receptors (MPRs) provided the first evidence for a direct interaction between GGAs and cargo (Bonifacino, 2004). The key residues in the C-tails of the cation-dependent MPR (CD-MPR), cation-independent MPR (CI-MPR), sortilin, memapsin 2 (β-secretase [BACE]), and low-density-lipoprotein receptor-related protein 3 for binding to the VHS domain of human GGAs were shown to constitute acidic dileucine motifs with the consensus sequence DXXLL (Nielsen et al, 2001;Puertollano et al, 2001;Takatsu et al, 2001;Zhu et al, 2001;He et al, 2002;Misra et al, 2002). Phosphorylation of a Ser residue within or adjacent to the acidic dileucine motif enhanced the binding of CI-MPR, BACE, sorLA, and sortilin C-tail sequences to human GGAs, suggesting a phosphoregulated mechanism for binding and cargo sorting He et al, 2003;Cramer et al, 2010).…”