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

Umasankar, Perunthottathu K; Ma, Li; Thieman, J. R.; Jha, Anupma; Doray, Balraj; Watkins, Simon C.; Traub, Linton M.

doi:10.7554/elife.04137

Cited by 63 publications

(135 citation statements)

References 115 publications

(248 reference statements)

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“…Without clathrin, clathrin-mediated trafficking is blocked. In the absence of FCHO2, endocytosis still progresses, yet clathrin-coated pits are abnormally arranged, with AP-2-positive structures appearing enlarged and clustered [37]. We assessed what the impact of disrupting these genes has on the intracellular replication and CCV formation of Coxiella .…”

Section: Resultsmentioning

confidence: 99%

“…FCHO2 belongs to the muniscin family of proteins, which also includes FCHO1 and SGIP1 [40]. FCHO1 and 2 are thought to be involved in initiating clathrin-mediated endocytosis, though this is debated in the field as clathrin-mediated endocytosis still occurs in the host in the absence of FCHO1/2 albeit with abnormal morphology [33, 37, 41]. Nevertheless, FCHO2 arrives early to the site of clathrin-mediated endocytosis and aids in sculpting the plasma membrane to form the spherical clathrin-coated vesicles [32, 34].…”

Section: Discussionmentioning

confidence: 99%

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The Effector Cig57 Hijacks FCHO-Mediated Vesicular Trafficking to Facilitate Intracellular Replication of Coxiella burnetii

et al. 2016

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Coxiella burnetii is an intracellular bacterial pathogen that infects alveolar macrophages and replicates within a unique lysosome-derived vacuole. When Coxiella is trafficked to a host cell lysosome the essential Dot/Icm type IV secretion system is activated allowing over 130 bacterial effector proteins to be translocated into the host cytosol. This cohort of effectors is believed to manipulate host cell functions to facilitate Coxiella-containing vacuole (CCV) biogenesis and bacterial replication. Transposon mutagenesis has demonstrated that the Dot/Icm effector Cig57 is required for CCV development and intracellular replication of Coxiella. Here, we demonstrate a role for Cig57 in subverting clathrin-mediated traffic through its interaction with FCHO2, an accessory protein of clathrin coated pits. A yeast two-hybrid screen identified FCHO2 as a binding partner of Cig57 and this interaction was confirmed during infection using immunoprecipitation experiments. The interaction between Cig57 and FCHO2 is dependent on one of three endocytic sorting motif encoded by Cig57. Importantly, complementation analysis demonstrated that this endocytic sorting motif is required for full function of Cig57. Consistent with the intracellular growth defect in cig57-disrupted Coxiella, siRNA gene silencing of FCHO2 or clathrin (CLTC) inhibits Coxiella growth and CCV biogenesis. Clathrin is recruited to the replicative CCV in a manner that is dependent on the interaction between Cig57 and FCHO2. Creation of an FCHO2 knockout cell line confirmed the importance of this protein for CCV expansion, intracellular replication of Coxiella and clathrin recruitment to the CCV. Collectively, these results reveal Cig57 to be a significant virulence factor that co-opts clathrin-mediated trafficking, via interaction with FCHO2, to facilitate the biogenesis of the fusogenic Coxiella replicative vacuole and enable intracellular success of this human pathogen.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

The Effector Cig57 Hijacks FCHO-Mediated Vesicular Trafficking to Facilitate Intracellular Replication of Coxiella burnetii

et al. 2016

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“…Further, the proteins containing those motifs are cytosolic coat assembly/disassembly factors rather than membrane-embedded cargo, and as such δ-COP MHDs may play a role more analogous to that of the MHDs of muniscin family members such as Syp1p and its mammalian homologs FCHo1 and FCHo2 (39), than to the role of the MHDs of AP complexes in clathrin-coated vesicle formation.…”

Section: Discussionmentioning

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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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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“…5 However, single-molecule imaging experiments suggest that FCHO1/2 "stabilize" the growing bud but do not initiate curvature. 46 Efficient recruitment of binding partners and activation of AP2, 43,44 aided by clustering from oligomerization, 5 may instead explain how FCHO2 acts as a nucleator of clathrin-mediated endocytosis. Indeed, multiple other proteins at endocytic sites are likely responsible for curvature generation, including the FCHO2 binding partner Eps15, 47,48 multiple classical BAR domain proteins, 33 and the triskelion clathrin coat.…”

Section: Scaffolding Functionsmentioning

confidence: 99%

“…43,44 In yeast, Syp1 acts similarly; it is present early at sites of endocytosis and recruits Ede1, an Eps15 homolog. 14,45 The tubulating activity of FCHO1/2 F-BAR domains 3 led to the idea that they might induce the initial membrane curvature early at an endocytic site.…”

Section: Scaffolding Functionsmentioning

confidence: 99%

Linking up at the BAR: Oligomerization and F-BAR protein function

McDonald

Gould

2016

Cell Cycle

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As cells grow, move, and divide, they must reorganize and rearrange their membranes and cytoskeleton. The F-BAR protein family links cellular membranes with actin cytoskeletal rearrangements in processes including endocytosis, cytokinesis, and cell motility. Here we review emerging information on mechanisms of F-BAR domain oligomerization and membrane binding, and how these activities are coordinated with additional domains to accomplish scaffolding and signaling functions.

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

Cited by 63 publications

References 115 publications

The Effector Cig57 Hijacks FCHO-Mediated Vesicular Trafficking to Facilitate Intracellular Replication of Coxiella burnetii

The Effector Cig57 Hijacks FCHO-Mediated Vesicular Trafficking to Facilitate Intracellular Replication of Coxiella burnetii

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

Linking up at the BAR: Oligomerization and F-BAR protein function

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