Ubiquitination drives COPI priming and Golgi SNARE localization

Deciphering mechanisms controlling SNARE localization within the Golgi complex is crucial to understanding protein trafficking patterns within the secretory pathway. SNAREs are also thought to prime COPI assembly to ensure incorporation of these essential cargoes into vesicles but the regulation of these events is poorly understood. Here we report roles for ubiquitin recognition by COPI in SNARE trafficking and in stabilizing interactions between Arf, COPI, and Golgi SNAREs. The ability of COPI to bind ubiquitin through its N-terminal WD repeat domain of β′COP or through an unrelated ubiquitin-binding domain (UBD) is essential for the proper localization of Golgi SNAREs Bet1 and Gos1. We find that COPI, the ArfGAP Glo3, and multiple Golgi SNAREs are ubiquitinated. Notably, the binding of Arf and COPI to Gos1 is markedly enhanced by ubiquitination of these components. Glo3 is thought to prime COPI-SNARE interactions; however, Glo3 is not enriched in the ubiquitin-stabilized SNARE-Arf-COPI complex but is instead enriched with COPI complexes that lack SNAREs. These results support a new model for how posttranslational modifications drive COPI priming events crucial for Golgi SNARE localization.

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Ubiquitin Signals Protein Trafficking via Interaction with a Novel Ubiquitin Binding Domain in the Membrane Fusion Regulator, Vps9p

Current Biology ◽

10.1016/s0960-9822(03)00043-5 ◽

2003 ◽

Vol 13 (3) ◽

pp. 258-262 ◽

Cited By ~ 77

Author(s):

Kathryn M. Donaldson ◽

Hongwei Yin ◽

Nicholas Gekakis ◽

Frantisek Supek ◽

Claudio A.P. Joazeiro

Keyword(s):

Membrane Fusion ◽

Protein Trafficking ◽

Binding Domain ◽

Ubiquitin Binding ◽

Ubiquitin Binding Domain

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Faculty Opinions recommendation of The ubiquitin binding domain ZnF UBP recognizes the C-terminal diglycine motif of unanchored ubiquitin.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1030611.370788 ◽

2006 ◽

Author(s):

Jane Endicott

Keyword(s):

Binding Domain ◽

Ubiquitin Binding ◽

Ubiquitin Binding Domain

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A20 prevents inflammasome-dependent arthritis by inhibiting macrophage necroptosis through its ZnF7 ubiquitin-binding domain

Nature Cell Biology ◽

10.1038/s41556-019-0324-3 ◽

2019 ◽

Vol 21 (6) ◽

pp. 731-742 ◽

Cited By ~ 40

Author(s):

Apostolos Polykratis ◽

Arne Martens ◽

Remzi Onur Eren ◽

Yoshitaka Shirasaki ◽

Mai Yamagishi ◽

...

Keyword(s):

Binding Domain ◽

Ubiquitin Binding ◽

Ubiquitin Binding Domain

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CoCUN, a Novel Ubiquitin Binding Domain Identified in N4BP1

Biomolecules ◽

10.3390/biom9070284 ◽

2019 ◽

Vol 9 (7) ◽

pp. 284 ◽

Cited By ~ 5

Author(s):

Ridvan Nepravishta ◽

Federica Ferrentino ◽

Walter Mandaliti ◽

Anna Mattioni ◽

Janine Weber ◽

...

Keyword(s):

Molecular Mechanisms ◽

Structural Model ◽

Functional Characterization ◽

Binding Domain ◽

15N Nmr ◽

Protein Sequence Analysis ◽

Hydrophobic Patch ◽

Binding Domains ◽

Ubiquitin Binding ◽

Ubiquitin Binding Domain

Ubiquitin binding domains (UBDs) are modular elements that bind non-covalently to ubiquitin and act as downstream effectors and amplifiers of the ubiquitination signal. With few exceptions, UBDs recognize the hydrophobic path centered on Ile44, including residues Leu8, Ile44, His68, and Val70. A variety of different orientations, which can be attributed to specific contacts between each UBD and surface residues surrounding the hydrophobic patch, specify how each class of UBD specifically contacts ubiquitin. Here, we describe the structural model of a novel ubiquitin-binding domain that we identified in NEDD4 binding protein 1 (N4BP1). By performing protein sequence analysis, mutagenesis, and nuclear magnetic resonance (NMR) spectroscopy of the 15N isotopically labeled protein, we demonstrate that a Phe-Pro motif in N4BP1 recognizes the canonical hydrophobic patch of ubiquitin. This recognition mode resembles the molecular mechanism evolved in the coupling of ubiquitin conjugation to endoplasmic-reticulum (ER) degradation (CUE) domain family, where an invariant proline, usually following a phenylalanine, is required for ubiquitin binding. Interestingly, this novel UBD, which is not evolutionary related to CUE domains, shares a 40% identity and 47% similarity with cullin binding domain associating with NEDD8 (CUBAN), a protein module that also recognizes the ubiquitin-like NEDD8. Based on these features, we dubbed the region spanning the C-terminal 50 residues of N4BP1 the CoCUN domain, for Cousin of CUBAN. By performing circular dichroism and 15N NMR chemical shift perturbation of N4BP1 in complex with ubiquitin, we demonstrate that the CoCUN domain lacks the NEDD8 binding properties observed in CUBAN. We also show that, in addition to mediating the interaction with ubiquitin and ubiquitinated substrates, both CUBAN and CoCUN are poly-ubiquitinated in cells. The structural and the functional characterization of this novel UBD can contribute to a deeper understanding of the molecular mechanisms governing N4BP1 function, providing at the same time a valuable tool for clarifying how the discrimination between ubiquitin and the highly related NEDD8 is achieved.

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