ULK complex organization in autophagy by a C-shaped FIP200 N-terminal domain dimer

Shi, Xiaoshan; Yokom, Adam L.; Wang, Chunxin; Young, L. C. T.; Youle, Richard J.; Hurley, James H.

doi:10.1101/840009

Cited by 21 publications

(31 citation statements)

References 52 publications

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“…Purified ULK1 complex was active with respect to the synthetic substrate, ULKtide ( Figure S2A). Difference maps for H/D exchange showed a clear protection pattern in the FIP200NTD, similar to our previous results on the FIP200 NTD alone (Shi et al, 2020). Specifically, peptides spanning residues 75-80, 157-165, 313-324, 350-356, 435-469 and 532-541 showed increases in protection of >10% after exchanging for 60 s ( Figure 1C).…”

Section: Full Length Fip200 Forms An Elongated Scaffold For Ulk1 Andsupporting

confidence: 89%

“…After affinity purification, full-length GST-FIP200-MBP was characterized using negative stain electron microscopy (NSEM) ( Figure 1A, Figure S1A). Full length FIP200 single particles showed an extended density with a C-shaped density at one end, corresponding to the dimeric FIP200NTD previously resolved (Shi et al, 2020). Density for the GST tag can be seen in the center of the FIP200 NTD dimer ( Figure S1B).…”

Section: Full Length Fip200 Forms An Elongated Scaffold For Ulk1 Andmentioning

confidence: 85%

“…Despite progress in crystallizing substructures of the ULK1 complex (Lin & Hurley, 2016) and an intermediate resolution cryo-EM structure of the FIP200 NTD (Shi et al, 2020), progress on the intact ULK1 complex containing full-length FIP200 has been very challenging.…”

Section: Introductionmentioning

confidence: 99%

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The autophagy adaptor NDP52 and the FIP200 coiled-coil allosterically activate ULK1 complex membrane recruitment

Shi

Chang

Yokom

et al. 2020

Preprint

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The selective autophagy pathways of xenophagy and mitophagy are initiated when the adaptor NDP52 recruits the ULK1 complex to autophagic cargo. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) was used to map the membrane and NDP52 binding sites of the ULK1 complex to unique regions of the coiled coil of the FIP200 subunit. Electron microscopy of the full-length ULK1 complex shows that the FIP200 coiled coil projects away from the crescent-shaped FIP200 N-terminal domain dimer. NDP52 allosterically stimulates membrane-binding by FIP200 and the ULK1 complex by promoting a more dynamic conformation of the membrane-binding portion of the FIP200 coiled coil. Giant unilamellar vesicle (GUV) reconstitution confirmed that membrane recruitment by the ULK1 complex is triggered by NDP52 engagement. These data reveal how the allosteric linkage between NDP52 and the ULK1 complex could drive the first membrane recruitment event of phagophore biogenesis in xenophagy and mitophagy.

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Section: Full Length Fip200 Forms An Elongated Scaffold For Ulk1 Andsupporting

confidence: 89%

Section: Full Length Fip200 Forms An Elongated Scaffold For Ulk1 Andmentioning

confidence: 85%

See 1 more Smart Citation

The autophagy adaptor NDP52 and the FIP200 coiled-coil allosterically activate ULK1 complex membrane recruitment

Shi

Chang

Yokom

et al. 2020

Preprint

Self Cite

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“…crYOLO [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22] . For example, Pang et al 23 used crYOLO to selectively pick particles, which were attached to liposomes; Rogala et al 14 highlighted in their study about mTORC1 that crYOLO was especially useful to exclude particles on carbon; Joppe et al 24 made use of crYOLO in a streamlined pipeline for rapid structure determination of yeast fatty acid synthase; and the new filament mode was recently used by Pospich et al to examine the structural effects of toxins on actin filaments 16 .…”

Section: Impact Of Cryolomentioning

confidence: 99%

The evolution of SPHIRE-crYOLO particle picking and its application in automated cryo-EM processing workflows

Wagner

Raunser

2020

Commun Biol

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Particle selection is a crucial step when processing electron cryo microscopy data. Several automated particle picking procedures were developed in the past but most struggle with non-ideal data sets. In our recent Communications Biology article, we presented crYOLO, a deep learning based particle picking program. It enables fast, automated particle picking at human levels of accuracy with low effort. A general model allows the use of crYOLO for selecting particles in previously unseen data sets without further training. Here we describe how crYOLO has evolved since its initial release. We have introduced filament picking, a new denoising technique, and a new graphical user interface. Moreover, we outline its usage in automated processing pipelines, which is an important advancement on the horizon of the field.The crYOLO particle picking procedure A major goal of electron cryo microscopy (cryo-EM) is to obtain high-resolution threedimensional (3D) reconstructions of proteins and protein complexes to gain novel biological insights. This process involves the selection of thousands to millions of noisy two-dimensional (2D) particle projections, a number that only keeps increasing with recent advances in hardware and software development.In our recent work in Communications Biology 1 we introduced the "crYOLO" particle picking procedure. It is based on a deep neural network and the You Only Look Once (YOLO) object detection framework 2 . This approach enables the automated picking of particles within cryo-EM micrographs with a low signal-to-noise ratio requiring minimal human supervision or intervention. CrYOLO is easy to configure and train on a specific data set. It is fast and can process up to six micrographs per second. As crYOLO sees the complete micrograph, it is able to learn the

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“…Evidence suggests that FIP200 is a functional homolog to the Atg1 complex scaffold subunits Atg11 and Atg17. Notably, Atg11, Atg17, and FIP200 are able to form a dimer [5][6][7].…”

Section: Autophagy Initiation and Phosphorylationmentioning

confidence: 99%

Mitotic phosphorylation of the ULK complex regulates cell cycle progression

2020

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Autophagy is an intracellular degradation pathway targeting organelles and macromolecules, thereby regulating various cellular functions. Phosphorylation is a key posttranscriptional protein modification implicated in the regulation of biological function including autophagy. Under asynchronous conditions, autophagy activity is predominantly suppressed by mechanistic target of rapamycin (mTOR) kinase, but whether autophagy-related genes (ATG) proteins are phosphorylated differentially throughout the sequential phases of the cell cycle remains unclear. In this issue, Li and colleagues report that cyclin-dependent kinase 1 (CDK1) phosphorylates the ULK complex during mitosis. This phosphorylation induces autophagy and, surprisingly, is shown to drive cell cycle progression. This work reveals a yet-unappreciated role for autophagy in cell cycle progression and enhances our understanding of the specific phase-dependent autophagy regulation during cellular growth and proliferation.

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ULK complex organization in autophagy by a C-shaped FIP200 N-terminal domain dimer

Cited by 21 publications

References 52 publications

The autophagy adaptor NDP52 and the FIP200 coiled-coil allosterically activate ULK1 complex membrane recruitment

The autophagy adaptor NDP52 and the FIP200 coiled-coil allosterically activate ULK1 complex membrane recruitment

The evolution of SPHIRE-crYOLO particle picking and its application in automated cryo-EM processing workflows

Mitotic phosphorylation of the ULK complex regulates cell cycle progression

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