Self-assembly and structure of a clathrin-independent AP-1:Arf1 tubular membrane coat

Hooy, Richard M.; Iwamoto, Yuichiro; Tudorica, Dan A.; Ren, Xuefeng; Hurley, James H.

doi:10.1126/sciadv.add3914

Cited by 11 publications

(10 citation statements)

References 77 publications

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“…AP-1 15 , 16 , 33 , AP-3 18 , 34 , and AP-4 35 have all been reported to be recruited to membranes through direct interaction with the small GTPase, Arf1. Through a multitude of structures and mutational analyses 26 , 27 , AP-1 has been shown to bind to Arf1 in two distinct locations — the γ and β1 subunits. Each interface interacts with Arf1 through the Switch I and II regions, explaining the GTP dependence of Arf1/AP-1 binding.…”

Section: Resultsmentioning

confidence: 99%

“…The insight that AP-1 is natively closed 10 and AP-3 is natively open suggests that each complex has distinct pathways to forming vesicular and tubular coats. A recent report showed that AP-1 can polymerize on membrane tubules in an Arf1-dependent, clathrin-independent manner 26 , suggesting that AP-1 acts to deform membranes and sort cargo into tubular assemblies before budding into clathrin-coated structures. However, a similar understanding of AP-3 function is hampered by a lack of structural and mechanistic information.…”

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confidence: 99%

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A structure-based mechanism for initiation of AP-3 coated vesicle formation

Begley,

Aragon,

Baker

2024

Preprint

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Adaptor protein complex 3 (AP-3) mediates cargo sorting from endosomes to lysosomes and lysosome-related organelles. Recently, it was shown that AP-3 is in a constitutively open, active conformation compared to the related AP-1 and AP-2 coat complexes, which are inactive until undergoing large conformational changes upon membrane recruitment. How AP-3 is regulated is therefore an open question. To understand the mechanism of AP-3 membrane recruitment and activation, we reconstituted the core of human AP-3 and determined multiple structures in the soluble and membrane-bound states using electron cryo-microscopy (cryo-EM). Similar to yeast AP-3, human AP-3 is in a constitutively open conformation, with the cargo-binding domain of the μ3 subunit conformationally free. To reconstitute AP-3 activation by the small GTPase Arf1, we used lipid nanodiscs to build Arf1-AP-3 complexes on membranes and determined three structures that show the stepwise conformational changes required for formation of AP-3 coated vesicles. First, membrane-recruitment is driven by one of two predicted Arf1 binding sites on AP-3. In this conformation, AP-3 is flexibly tethered to the membrane and its cargo binding domain remains conformationally dynamic. Second, cargo binding causes AP-3 to adopt a fixed position and rigidifies the complex, which stabilizes binding for a second Arf1 molecule. Finally, binding of the second Arf1 molecule provides the template for AP-3 dimerization, providing a glimpse into the first step of coat polymerization. We propose coat polymerization only occurs after cargo engagement, thereby linking cargo sorting with assembly of higher order coat structures. Additionally, we provide evidence for two amphipathic helices in AP-3, suggesting that AP-3 contributes to membrane deformation during coat assembly. In total, these data provide evidence for the first stages of AP-3 mediated vesicle coat assembly.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

A structure-based mechanism for initiation of AP-3 coated vesicle formation

Begley,

Aragon,

Baker

2024

Preprint

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“…6D-E). Similarly, aside from its ability to induce membrane curvature, clathrin might be required for the clustering of AP-1 at sites of cargo exchange, as the lack of clathrin leads to diffused localization of AP-1 along tubular structures 55 . The presence of AP-1 and AP-3 nanodomains on ARF1 compartments involved in Tfn recycling (Extended Data Fig.…”

Section: Discussionmentioning

confidence: 99%

ARF1 compartments direct cargo flow via maturation into recycling endosomes

Stockhammer,

Adarska,

Natalia

et al. 2023

Preprint

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Cellular membrane homeostasis is maintained via a tightly regulated membrane and cargo flow between organelles of the endocytic and secretory pathways. Adaptor protein complexes (APs), which are recruited to membranes by the small GTPase ARF1, facilitate cargo selection and incorporation into trafficking intermediates. According to the classical model, small vesicles would facilitate bi-directional long-range vesicular transport between the Golgi and endosomes. Here we revisit the intracellular organization of the vesicular sorting machinery using a combination of CRISPR-Cas9 gene editing, live-cell high temporal (fast-confocal) or spatial (stimulated emission depletion (STED)) microscopy as well as correlative light and electron microscopy. We characterize novel tubulo-vesicular ARF1 compartments that harbor clathrin and different APs, and coordinate Golgi export as well as endocytic recycling. Rather than long-range vesicle shuttling, we observe transient interactions of ARF1 compartments and recycling endosomes, with the sorting machinery localizing at the interface between the two membranes. Our findings suggest that endocytic and secretory cargo transfer from ARF1 compartments to endosomes may be mediated by a kiss-and-run mechanism rather than by fusion of transport vesicles.

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“…Ion-pair reversed-phase acetonitrile gradient HPLC was used to assay the nucleotide bound to VPS15 with a protocol adapted from ref. 35 . First, PI3KC3-C1 was denatured by heating in a heat block at 90 C for 10 minutes.…”

Section: Methodsmentioning

confidence: 99%

Structural pathway for class III PI 3-kinase activation by the myristoylated GTP-binding pseudokinase VPS15

Cook,

Chen,

Ren

et al. 2023

Preprint

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The class III phosphatidylinositol (PI) 3-kinase complexes I and II (PI3KC3-C1 and -C2) are central to the initiation of macroautophagy and endosomal maturation, respectively. Through three-dimensional classification of a large cryo-EM dataset of human PI3KC3-C1 bound to the small GTPase RAB1A, we were able to map the structural pathway of enzyme activation. The inactive conformation is stabilized by anN-myristoyl modification of the pseudokinase (PK) subunit VPS15. TheN-myristate is sequestered in the N-lobe of the VPS15 PK domain, which stabilizes a series of interactions whereby VPS15 sequesters and blocks the catalytic and membrane binding units of the VPS34 lipid kinase. In the activated conformation, theN-myristate and the VPS34 lipid kinase domain are liberated to interact with membranes and catalyze PI3P formation. The VPS15 PK domain contains a unique Arg at the gatekeeper position and binds tightly to GTP. GTP binding structurally stabilizes theN-myristate “in” conformation, which promotes the inactive conformation. This pathway provides a general mechanism for PI3KC3 activation in autophagy and endosome biogenesis and a roadmap for their pharmacological upregulation.

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Self-assembly and structure of a clathrin-independent AP-1:Arf1 tubular membrane coat

Cited by 11 publications

References 77 publications

A structure-based mechanism for initiation of AP-3 coated vesicle formation

A structure-based mechanism for initiation of AP-3 coated vesicle formation

ARF1 compartments direct cargo flow via maturation into recycling endosomes

Structural pathway for class III PI 3-kinase activation by the myristoylated GTP-binding pseudokinase VPS15

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