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9A structure of the COPI coat reveals that the Arf1 GTPase occupies two contrasting molecular environments.

ABSTRACT: COPI coated vesicles mediate trafficking within the Golgi apparatus and between the Golgi and the endoplasmic reticulum. Assembly of a COPI coated vesicle is initiated by the small GTPase Arf1 that recruits the coatomer complex to the membrane, triggering polymerization and budding. The vesicle uncoats before fusion with a target membrane. Coat components are structurally conserved between COPI and clathrin/adaptor proteins. Using cryo-electron tomography and subtomogram averaging, we determined the structure of the COPI coat assembled on membranes in vitro at 9 Å resolution. We also obtained a 2.57 Å resolution crystal structure of ??-COP. By combining these structures we built a molecular model of the coat. We additionally determined the coat structure in the presence of ArfGAP proteins that regulate coat dissociation. We found that Arf1 occupies contrasting molecular environments within the coat, leading us to hypothesize that some Arf1 molecules may regulate vesicle assembly while others regulate coat disassembly.

SUBMITTER: Dodonova SO

PROVIDER: S-EPMC5482573 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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9Å structure of the COPI coat reveals that the Arf1 GTPase occupies two contrasting molecular environments.

Dodonova Svetlana O SO Aderhold Patrick P Kopp Juergen J Ganeva Iva I Röhling Simone S Hagen Wim J H WJH Sinning Irmgard I Wieland Felix F Briggs John A G JAG

eLife 20170616

COPI coated vesicles mediate trafficking within the Golgi apparatus and between the Golgi and the endoplasmic reticulum. Assembly of a COPI coated vesicle is initiated by the small GTPase Arf1 that recruits the coatomer complex to the membrane, triggering polymerization and budding. The vesicle uncoats before fusion with a target membrane. Coat components are structurally conserved between COPI and clathrin/adaptor proteins. Using cryo-electron tomography and subtomogram averaging, we determined ...[more]

PMID: 28621666

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Project description:Hazara nairovirus (HAZV) is an enveloped trisegmented negative-strand RNA virus classified within the Nairoviridae family of the Bunyavirales order and a member of the same subtype as Crimean-Congo hemorrhagic fever virus, responsible for fatal human disease. Nairoviral subversion of cellular trafficking pathways to permit viral entry, gene expression, assembly, and egress is poorly understood. Here, we generated a recombinant HAZV expressing enhanced green fluorescent protein and used live-cell fluorescent imaging to screen an siRNA library targeting genes involved in cellular trafficking networks, the first such screen for a nairovirus. The screen revealed prominent roles for subunits of the coat protein 1 (COPI)-vesicle coatomer, which regulates retrograde trafficking of cargo between the Golgi apparatus and the endoplasmic reticulum, as well as intra-Golgi transport. We show the requirement of COPI-coatomer subunits impacted at least two stages of the HAZV replication cycle: an early stage prior to and including gene expression and also a later stage during assembly and egress of infectious virus, with COPI-knockdown reducing titers by approximately 1,000-fold. Treatment of HAZV-infected cells with brefeldin A (BFA), an inhibitor of Arf1 activation required for COPI coatomer formation, revealed that this late COPI-dependent stage was Arf1 dependent, consistent with the established role of Arf1 in COPI vesicle formation. In contrast, the early COPI-dependent stage was Arf1 independent, with neither BFA treatment nor siRNA-mediated ARF1 knockdown affecting HAZV gene expression. HAZV exploitation of COPI components in a noncanonical Arf1-independent process suggests that COPI coatomer components may perform roles unrelated to vesicle formation, adding further complexity to our understanding of cargo-mediated transport.IMPORTANCE Nairoviruses are tick-borne enveloped RNA viruses that include several pathogens responsible for fatal disease in humans and animals. Here, we analyzed host genes involved in trafficking networks to examine their involvement in nairovirus replication. We revealed important roles for genes that express multiple components of the COPI complex, which regulates transport of Golgi apparatus-resident cargos. COPI components influenced at least two stages of the nairovirus replication cycle: an early stage prior to and including gene expression and also a later stage during assembly of infectious virus, with COPI knockdown reducing titers by approximately 1,000-fold. Importantly, while the late stage was Arf1 dependent, as expected for canonical COPI vesicle formation, the early stage was found to be Arf1 independent, suggestive of a previously unreported function of COPI unrelated to vesicle formation. Collectively, these data improve our understanding of nairovirus host-pathogen interactions and suggest a new Arf1-independent role for components of the COPI coatomer complex.

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9A structure of the COPI coat reveals that the Arf1 GTPase occupies two contrasting molecular environments.

Publications

9Å structure of the COPI coat reveals that the Arf1 GTPase occupies two contrasting molecular environments.

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