Balraj Doray

Autoinhibition of the ligand-binding site of GGA1/3 VHS domains by an internal acidic cluster-dileucine motif.

The GGAs (Golgi-localizing, γ-adaptin ear homology domain, ARF-binding proteins) are a family of proteins implicated in protein trafficking from the Golgi to endosomes/lysosomes. These proteins have modular structures with an N-terminal VHS (VPS-27, Hrs, and STAM) domain followed by a GAT (GGA and TOM1) domain, a connecting hinge segment, and a C-terminal GAE (γ-adaptin ear) domain. Isolated VHS domains have been shown to bind specifically to acidic cluster (AC)-dileucine motifs present in the cytoplasmic tails of the mannose 6-phosphate receptors. Here we report that full-length cytoplasmic GGA1 and GGA3 but not GGA2 bind the cation-independent mannose 6-phosphate receptor very poorly because of autoinhibition. This inhibition is caused by the binding of an AC-LL sequence present in the hinge segment to the ligand-binding site in the VHS domain. The inhibition depends on the phosphorylation of a serine located three residues upstream of the AC-LL motif. The serine is phosphorylated by casein kinase 2 in in vitro assays. Substitution of the GGA1 inhibitory sequence into the analogous location in GGA2, which lacks the AC-LL motif, results in autoinhibition of the latter protein. These data indicate that the activity of GGA1 and GGA3 is regulated by cycles of phosphorylation/dephosphorylation.

Clathrin Regulates the Association of PIPKIγ661 with the AP-2 Adaptor β2 Appendage

The AP-2 clathrin adaptor differs fundamentally from the related AP-1, AP-3, and AP-4 sorting complexes because membrane deposition does not depend directly on an Arf family GTPase. Instead phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) appears to act as the principal compartmental cue for AP-2 placement at the plasma membrane as well as for the docking of numerous other important clathrin coat components at the nascent bud site. This PtdIns(4,5)P2 dependence makes type I phosphatidylinositol 4-phosphate 5-kinases (PIPKIs) lynchpin enzymes in the assembly of clathrin-coated structures at the cell surface. PIPKIγ is the chief 5-kinase at nerve terminals, and here we show that the 26-amino acid, alternatively spliced C terminus of PIPKIγ661 is an intrinsically unstructured polypeptide that binds directly to the sandwich subdomain of the AP-2 β2 subunit appendage. An aromatic side chain-based, extended interaction motif that also includes the two bulky C-terminal residues of the short PIPKIγ635 variant is necessary for β2 appendage engagement. The clathrin heavy chain accesses the same contact surface on the AP-2 β2 appendage, but because of additional clathrin binding sites located within the unstructured hinge segment of the β2 subunit, clathrin binds the β2 chain with a higher apparent affinity than PIPKIγ661. A clathrin-regulated interaction with AP-2 could allow PIPKIγ661 to be strategically positioned for regional PtdIns(4,5)P2 generation during clathrin-coated vesicle assembly at the synapse.

Binding of cargo sorting signals to AP-1 enhances its association with ADP ribosylation factor 1–GTP

The adaptor protein AP-1 is the major coat protein involved in the formation of clathrin-coated vesicles at the trans-Golgi network. The prevailing view is that AP-1 recruitment involves coincident binding to multiple low-affinity sites comprising adenosine diphosphate ribosylation factor 1 (Arf-1)–guanosine triphosphate (GTP), cargo sorting signals, and phosphoinositides. We now show that binding of cargo signal peptides to AP-1 induces a conformational change in its core domain that greatly enhances its interaction with Arf-1–GTP. In addition, we provide evidence for cross talk between the dileucine and tyrosine binding sites within the AP-1 core domain such that binding of a cargo signal to one site facilitates binding to the other site. The stable association of AP-1 with Arf-1–GTP, which is induced by cargo signals, would serve to provide sufficient time for adaptor polymerization and clathrin recruitment while ensuring the packaging of cargo molecules into the forming transport vesicles.

Identification of Acidic Dileucine Signals in LRP9 that Interact with Both GGAs and AP‐1/AP‐2

The Golgi‐localized, gamma‐ear‐containing, ADP ribosylation factor‐binding family of monomeric clathrin adaptors (GGAs) is known to bind cargo molecules through short C‐terminal peptide motifs conforming to the sequence DXXLL (X = any amino acid), while the heterotetrameric adaptors AP‐1 and AP‐2 utilize a similar but discrete sorting motif of the sequence [D,E]XXXL[L,I]. While it has been established that a single cargo molecule may contain either or both types of these acidic cluster‐dileucine (AC‐LL) sorting signals, there are no examples of cargo with overlapping GGA and AP‐1/AP‐2‐binding motifs. In this study, we report that the cytosolic tail of low‐density lipoprotein receptor‐related protein (LRP)9 contains a bifunctional GGA and AP‐1/AP‐2‐binding motif at its carboxy‐terminus (EDEPLL). We further demonstrate that the internal EDEVLL sequence of LRP9 also binds to GGAs in addition to AP‐2. Either AC‐LL motif of LRP9 is functional in endocytosis. These findings represent the first study characterizing the trafficking of LRP9 and also have implications for the identification of additional GGA cargo molecules.

A clathrin coat assembly role for the muniscin protein central linker revealed by TALEN-mediated gene editing

Clathrin-mediated endocytosis is an evolutionarily ancient membrane transport system regulating cellular receptivity and responsiveness. Plasmalemma clathrin-coated structures range from unitary domed assemblies to expansive planar constructions with internal or flanking invaginated buds. Precisely how these morphologically-distinct coats are formed, and whether all are functionally equivalent for selective cargo internalization is still disputed. We have disrupted the genes encoding a set of early arriving clathrin-coat constituents, FCHO1 and FCHO2, in HeLa cells. Endocytic coats do not disappear in this genetic background; rather clustered planar lattices predominate and endocytosis slows, but does not cease. The central linker of FCHO proteins acts as an allosteric regulator of the prime endocytic adaptor, AP-2. By loading AP-2 onto the plasma membrane, FCHO proteins provide a parallel pathway for AP-2 activation and clathrin-coat fabrication. Further, the steady-state morphology of clathrin-coated structures appears to be a manifestation of the availability of the muniscin linker during lattice polymerization. DOI: http://dx.doi.org/10.7554/eLife.04137.001

Multiple Domains of GlcNAc-1-phosphotransferase Mediate Recognition of Lysosomal Enzymes

The Golgi enzyme UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase), an α2β2γ2 hexamer, mediates the initial step in the addition of the mannose 6-phosphate targeting signal on newly synthesized lysosomal enzymes. This tag serves to direct the lysosomal enzymes to lysosomes. A key property of GlcNAc-1-phosphotransferase is its unique ability to distinguish the 60 or so lysosomal enzymes from the numerous non-lysosomal glycoproteins with identical Asn-linked glycans. In this study, we demonstrate that the two Notch repeat modules and the DNA methyltransferase-associated protein interaction domain of the α subunit are key components of this recognition process. Importantly, different combinations of these domains are involved in binding to individual lysosomal enzymes. This study also identifies the γ-binding site on the α subunit and demonstrates that in the majority of instances the mannose 6-phosphate receptor homology domain of the γ subunit is required for optimal phosphorylation. These findings serve to explain how GlcNAc-1-phosphotransferase recognizes a large number of proteins that lack a common structural motif.

Do GGA Adaptors Bind Internal DXXLL Motifs

The GGA family of clathrin adaptor proteins mediates the intracellular trafficking of transmembrane proteins by interacting with DXXLL‐type sorting signals on the latter. These signals were originally identified at the carboxy‐termini of the transmembrane cargo proteins. Subsequent studies, however, showed that internal DXXLL sorting motifs occur within the N‐ or C‐terminal cytoplasmic domains of cargo molecules. The GGAs themselves also contain internal DXXLL motifs that serve to auto‐regulate GGA function. A recent study challenged the notion that internal DXXLL signals are competent for binding to GGAs. Since the question of whether GGA adaptors interact with internal DXXLL motifs is fundamental to the identification of bona fide GGA cargo, and to an accurate understanding of GGA regulation within cells, we have extended our previous findings. We now present additional evidence confirming that GGAs do interact with internal DXXLL motifs. We also summarize the recent reports from other laboratories documenting internal GGA binding motifs.

Analysis of Gga null mice demonstrates a non-redundant role for mammalian GGA2 during development.

Numerous studies using cultured mammalian cells have shown that the three GGAs (Golgi-localized, gamma-ear containing, ADP-ribosylation factor- binding proteins) function in the transport of cargo proteins between the trans- Golgi network and endosomes. However, the in vivo role(s) of these adaptor proteins and their possible functional redundancy has not been analyzed. In this study, the genes encoding GGAs1-3 were disrupted in mice by insertional mutagenesis. Loss of GGA1 or GGA3 alone was well tolerated whereas the absence of GGA2 resulted in embryonic or neonatal lethality, depending on the genetic background of the mice. Thus, GGA2 mediates a vital function that cannot be compensated for by GGA1and/or GGA3. The combined loss of GGA1 and GGA3 also resulted in a high incidence of neonatal mortality but in this case the expression level of GGA2 may be inadequate to compensate for the loss of the other two GGAs. We conclude that the three mammalian GGAs are essential proteins that are not fully redundant.

Engineering of GlcNAc-1-Phosphotransferase for Production of Highly Phosphorylated Lysosomal Enzymes for Enzyme Replacement Therapy

Several lysosomal enzymes currently used for enzyme replacement therapy in patients with lysosomal storage diseases contain very low levels of mannose 6-phosphate, limiting their uptake via mannose 6-phosphate receptors on the surface of the deficient cells. These enzymes are produced at high levels by mammalian cells and depend on endogenous GlcNAc-1-phosphotransferase α/β precursor to phosphorylate the mannose residues on their glycan chains. We show that co-expression of an engineered truncated GlcNAc-1-phosphotransferase α/β precursor and the lysosomal enzyme of interest in the producing cells resulted in markedly increased phosphorylation and cellular uptake of the secreted lysosomal enzyme. This method also results in the production of highly phosphorylated acid β-glucocerebrosidase, a lysosomal enzyme that normally has just trace amounts of this modification.

Role of spacer‐1 in the maturation and function of GlcNAc‐1‐phosphotransferase

The UDP‐GlcNAc:lysosomal enzyme, N‐acetylglucosamine‐1‐phosphotransferase (GlcNAc‐1‐PT), is an α2β2γ2 hexamer that mediates the initial step in the formation of the mannose 6‐phosphate targeting signal on newly synthesized lysosomal acid hydrolases. The GNPTAB gene encodes the 1256 amino acid long α/β precursor which is normally cleaved at K928 in the early Golgi by Site‐1 protease (S1P). Here, we show that removal of the so‐called ‘spacer‐1′ domain (residues 86–322) results in cleavage almost exclusively at a second S1P consensus sequence located upstream of K928. In addition, GlcNAc‐1‐PT lacking spacer‐1 exhibits enhanced phosphorylation of several non‐lysosomal glycoproteins, while the phosphorylation of lysosomal acid hydrolases is not altered. In view of these effects on the maturation and function of GlcNAc‐1‐PT, we suggest renaming `spacer‐1′ the `regulatory‐1′ domain.