Structural Basis for the Recognition of Tyrosine-based Sorting Signals by the μ3A Subunit of the AP-3 Adaptor Complex

Mardones, Gonzalo A.; Burgos, Patricia V.; Lin, Yimo; Kloer, Daniel P.; Magadán, Javier G.; Hurley, James H.; Bonifacino, Juan S.

doi:10.1074/jbc.m113.450775

Cited by 44 publications

(54 citation statements)

References 49 publications

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“…1C) (16)(17)(18)(19)(20). One striking feature of the δ-COP MHD structure is its large negative electrostatic surface potential, which is conserved across species and therefore presumably is functionally important (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…1D). showing that di-tryptophan motifs bind to δ-COP at a position similar to that at which the YKFFE sequence of APP binds to the μ4 MHD (PDB ID code 3L81) (17) but at a completely different site from the binding of Yxxϕ signals to AP MHDs (PDB ID code 1BXX) (16,18,19). The final refined 2mF O -DF C density contoured at 1.2σ and mF O -DF C difference density at ±3.0σ for the peptide are shown in the Inset.…”

Section: Resultsmentioning

confidence: 99%

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Structural basis for the binding of tryptophan-based motifs by δ-COP

Suckling

Poon

Travis

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Coatomer consists of two subcomplexes: the membrane-targeting, ADP ribosylation factor 1 (Arf1):GTP-binding βγδζ-COP F-subcomplex, which is related to the adaptor protein (AP) clathrin adaptors, and the cargo-binding αβ'e-COP B-subcomplex. We present the structure of the C-terminal μ-homology domain of the yeast δ-COP subunit in complex with the WxW motif from its binding partner, the endoplasmic reticulum-localized Dsl1 tether. The motif binds at a site distinct from that used by the homologous AP μ subunits to bind YxxΦ cargo motifs with its two tryptophan residues sitting in compatible pockets. We also show that the Saccharomyces cerevisiae Arf GTPase-activating protein (GAP) homolog Gcs1p uses a related WxxF motif at its extreme C terminus to bind to δ-COP at the same site in the same way. Mutations designed on the basis of the structure in conjunction with isothermal titration calorimetry confirm the mode of binding and show that mammalian δ-COP binds related tryptophan-based motifs such as that from ArfGAP1 in a similar manner. We conclude that δ-COP subunits bind Wx n(1-6) [WF] motifs within unstructured regions of proteins that influence the lifecycle of COPI-coated vesicles; this conclusion is supported by the observation that, in the context of a sensitizing domain deletion in Dsl1p, mutating the tryptophan-based motif-binding site in yeast causes defects in both growth and carboxypeptidase Y trafficking/processing. C OPI vesicles mediate retrograde trafficking from the Golgi to the endoplasmic reticulum (ER) and within the Golgi (reviewed in refs. 1-3). The COPI vesicle coat consists of two major components, coatomer and the small GTPase ADP ribosylation factor 1 (Arf1). Coatomer is an ∼600 kDa heteroheptameric complex consisting of two linked subcomplexes, the βγδζ-COP F-subcomplex and the αβ'e-COP B-subcomplex, all conserved from yeast to humans.Recent studies have led to a model for COPI-coated vesicle formation in which the F-subcomplex functions as a traditional Arf1:GTP effector but with two membrane-attached Arf1:GTP molecules binding to quasi-equivalent sites on a single F-subcomplex, recruiting F-subcomplex and its associated B-subcomplex onto the membrane en bloc (4). Once the vesicle has budded from its donor membrane, an Arf GTPase-activating protein (ArfGAP) catalyzes the hydrolysis of GTP on Arf1, and the Arf1:GDP dissociates from the membrane, followed later by the dissociation of the coatomer complex that was bound to the transport vesicle (5). ArfGAPs also have been proposed to function at different stages in vesicle biogenesis, both as terminators and, during an earlier cargoediting step, as effectors (reviewed in depth in ref. 6). In the final stages of its life, a COPI-coated vesicle docks with the ER via the Dsl1-tethering complex, completes uncoating, and finally undergoes SNARE-mediated fusion with the ER (7-9).The two main ArfGAPs associated with COPI-dependent retrograde transport in yeast are Gcs1p and Glo3p. These proteins can substitute for one another, at least partially,...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Structural basis for the binding of tryptophan-based motifs by δ-COP

Suckling

Poon

Travis

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Each AP complex recognizes a subset of sorting motifs, and the variant amino acids within [DERQ]XXXL [LI] and YXXΦ sequences define the specificity of signal recognition (47)(48)(49). Disruption of AP complex interactions with sorting motifs can lead to missorting of cargo proteins back to the plasma membrane (50)(51)(52)(53).…”

Section: Discussionmentioning

confidence: 99%

Coxiella burnetii effector protein subverts clathrin-mediated vesicular trafficking for pathogen vacuole biogenesis

Larson

Beare

Howe

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

149

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Successful macrophage colonization by Coxiella burnetii, the cause of human Q fever, requires pathogen-directed biogenesis of a large, growth-permissive parasitophorous vacuole (PV) with phagolysosomal characteristics. The vesicular trafficking pathways co-opted by C. burnetii for PV development are poorly defined; however, it is predicted that effector proteins delivered to the cytosol by a defective in organelle trafficking/intracellular multiplication (Dot/ Icm) type 4B secretion system are required for membrane recruitment. Here, we describe involvement of clathrin-mediated vesicular trafficking in PV generation and the engagement of this pathway by the C. burnetii type 4B secretion system substrate Coxiella vacuolar protein A (CvpA). CvpA contains multiple dileucine [DERQ]XXXL [LI] and tyrosine (YXXΦ)-based endocytic sorting motifs like those recognized by the clathrin adaptor protein (AP) complexes AP1, AP2, and AP3. A C. burnetii ΔcvpA mutant exhibited significant defects in replication and PV development, confirming the importance of CvpA in infection. Ectopically expressed mCherry-CvpA localized to tubular and vesicular domains of pericentrosomal recycling endosomes positive for Rab11 and transferrin receptor, and CvpA membrane interactions were lost upon mutation of endocytic sorting motifs. Consistent with CvpA engagement of the endocytic recycling system, ectopic expression reduced uptake of transferrin. In pull-down assays, peptides containing CvpA-sorting motifs and full-length CvpA interacted with AP2 subunits and clathrin heavy chain. Furthermore, depletion of AP2 or clathrin by siRNA treatment significantly inhibited C. burnetii replication. Thus, our results reveal the importance of clathrincoated vesicle trafficking in C. burnetii infection and define a role for CvpA in subverting these transport mechanisms.type IV secretion | vesicular fusion

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“…This conformational change appears to involve pivoting on a third Arf1-binding site located on a different face of the g trunk (Ren et al 2013). Mardones et al 2013;Ren et al 2013), an endosome-associated adaptor involved in cargo sorting to lysosomes and lysosome-related organelles, indicating that this complex might function by a similar mechanism.…”

Section: Recognition Of Linear Motifs By Ap-2mentioning

confidence: 99%

Cargo Recognition in Clathrin-Mediated Endocytosis

Traub

Bonifacino

2013

Cold Spring Harbor Perspectives in Biology

279

290

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The endosomal system is expansive and complex, characterized by swift morphological transitions, dynamic remodeling of membrane constituents, and intracellular positioning changes. To properly navigate this ever-altering membrane labyrinth, transmembrane protein cargoes typically require specific sorting signals that are decoded by components of protein coats. The best-characterized sorting process within the endosomal system is the rapid internalization of select transmembrane proteins within clathrin-coated vesicles. Endocytic signals consist of linear motifs, conformational determinants, or covalent modifications in the cytosolic domains of transmembrane cargo. These signals are interpreted by a diverse set of clathrin-associated sorting proteins (CLASPs) that translocate from the cytosol to the inner face of the plasma membrane. Signal recognition by CLASPs is highly cooperative, involving additional interactions with phospholipids, Arf GTPases, other CLASPs, and clathrin, and is regulated by large conformational changes and covalent modifications. Related sorting events occur at other endosomal sorting stations.

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Structural Basis for the Recognition of Tyrosine-based Sorting Signals by the μ3A Subunit of the AP-3 Adaptor Complex

Cited by 44 publications

References 49 publications

Structural basis for the binding of tryptophan-based motifs by δ-COP

Structural basis for the binding of tryptophan-based motifs by δ-COP

Coxiella burnetii effector protein subverts clathrin-mediated vesicular trafficking for pathogen vacuole biogenesis

Cargo Recognition in Clathrin-Mediated Endocytosis

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