GGA Autoinhibition Revisited

Abstract

The cytosolic adaptors GGA1-3 mediate sorting of transmembrane proteins displaying a C-terminal acidic dileucine motif (DXXLL) in their cytosolic domain. GGA1 and GGA3 contain similar but intrinsic motifs that are believed to serve as autoinhibitory sites activated by the phosphorylation of a serine positioned three residues upstream of the DXXLL motif. In the present study, we have subjected the widely acknowledged concept of GGA1 autoinhibition to a thorough structural and functional examination. We find that (i) the intrinsic motif of GGA1 is inactive, (ii) only C-terminal DXXLL motifs constitute active GGA binding sites, (iii) while aspartates and phosphorylated serines one or two positions upstream of the DXXLL motif increase GGA1 binding, phosphoserines further upstream have little or no influence and (iv) phosphorylation of GGA1 does not affect its conformation or binding to Sortilin and SorLA. Taken together, our findings seem to refute the functional significance of GGA autoinhibition in particular and of intrinsic GGA binding motifs in general.

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

“…Thus, although recent work called into question the functional significance of autoinhibitory, VHS-binding sequences in the linker regions of mammalian GGA proteins (Cramer et al, 2010), an even more recent study challenged this view (Doray et al, 2012), and our data indicate that this is a property conserved in yeast Gga2p. Furthermore, despite the fact that the hinge regions are poorly conserved between yeast Gga1p and Gga2p (16% identity over 100 residues), the 380-395 deletion interval, which contains the internal Gga2p inhibitory sequence, contains a motif, DLLGD, that is conserved between the two proteins.…”

“…An alternative manual alignment of the Kex2p and Vps10p sequences with the mammalian GBS motifs aligns Asp residues in the yeast proteins with the conserved Asp of the mammalian GBS motifs and aligns at least one Leu in each yeast sequence with the conserved aliphatic residues of the mammalian GBS motifs. The crystal structures of these motifs bound to the VHS domain of GGA1 (sortilin, BACE, and SorLA) or GGA3 (CI-M6PR) identify key conserved residues in the mammalian VHS domains that are required for interaction with the Asp and Leu residues (or Asp, Val, and Met residues in the case of SorLA) of the GBS motifs Shiba et al, 2002;He et al, 2003;Cramer et al, 2010). As observed by Misra et al (2002), several of these VHS residues are not conserved in the yeast Gga1&2p VHS domains.…”

“…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). Comparison of the sequences of the yeast Gga proteins to the structures of the mammalian GGAs, however, suggested that the yeast proteins might not be able to bind the classic acidic dileucine motif .…”

Retromer Binds the FANSHY Sorting Motif in SorLA to Regulate Amyloid Precursor Protein Sorting and Processing

“…2 E). However, both proteins bound to the GGA1 VHS domain shown previously to interact in a region that shares no overlap with the FANSHY motif (Nielsen et al, 2001;Cramer et al, 2010) showing a specific disruption of the VPS26 binding site (Fig. 2 F).…”

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

“…This is consistent with a modulatory role for phosphorylation of Ser 780 , with phosphorylation facilitating Gga-dependent sorting. A modulatory role for phosphorylation would be consistent with the effects of phosphorylation of the C-tails of mammalian proteins on their interactions with mammalian Ggas (Supplemental Figure S9; Cramer et al, 2010).…”