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The open architecture of HD-PTP phosphatase provides new insights into the mechanism of regulation of ESCRT function.

ABSTRACT: HD-PTP is a tumour suppressor phosphatase that controls endocytosis, down-regulation of mitogenic receptors and cell migration. Central to its role is the specific recruitment of critical endosomal sorting complexes required for transport (ESCRTs). However, the molecular mechanisms that enable HD-PTP to regulate ESCRT function are unknown. We have characterised the molecular architecture of the entire ESCRT binding region of HD-PTP using small angle X-ray scattering and hydrodynamic analyses. We show that HD-PTP adopts an open and extended conformation, optimal for concomitant interactions with multiple ESCRTs, which contrasts with the compact conformation of the related ESCRT regulator Alix. We demonstrate that the HD-PTP open conformation is functionally competent for binding cellular protein partners. Our analyses rationalise the functional cooperation of HD-PTP with ESCRT-0, ESCRT-I and ESCRT-III and support a model for regulation of ESCRT function by displacement of ESCRT subunits, which is crucial in determining the fate of ubiquitinated cargo.

SUBMITTER: Gahloth D

PROVIDER: S-EPMC5567221 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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The open architecture of HD-PTP phosphatase provides new insights into the mechanism of regulation of ESCRT function.

Gahloth Deepankar D Heaven Graham G Jowitt Thomas A TA Mould A Paul AP Bella Jordi J Baldock Clair C Woodman Philip P Tabernero Lydia L

Scientific reports 20170822 1

HD-PTP is a tumour suppressor phosphatase that controls endocytosis, down-regulation of mitogenic receptors and cell migration. Central to its role is the specific recruitment of critical endosomal sorting complexes required for transport (ESCRTs). However, the molecular mechanisms that enable HD-PTP to regulate ESCRT function are unknown. We have characterised the molecular architecture of the entire ESCRT binding region of HD-PTP using small angle X-ray scattering and hydrodynamic analyses. We ...[more]

PMID: 28831121

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Project description:Although picornaviruses are conventionally considered 'nonenveloped', members of multiple picornaviral genera are released nonlytically from infected cells in extracellular vesicles. The mechanisms underlying this process are poorly understood. Here, we describe interactions of the hepatitis A virus (HAV) capsid with components of host endosomal sorting complexes required for transport (ESCRT) that play an essential role in release. We show release of quasi-enveloped virus (eHAV) in exosome-like vesicles requires a conserved export signal located within the 8 kDa C-terminal VP1 pX extension that functions in a manner analogous to late domains of canonical enveloped viruses. Fusing pX to a self-assembling engineered protein nanocage (EPN-pX) resulted in its ESCRT-dependent release in extracellular vesicles. Mutational analysis identified a 24 amino acid peptide sequence located within the center of pX that was both necessary and sufficient for nanocage release. Deleting a YxxL motif within this sequence ablated eHAV release, resulting in virus accumulating intracellularly. The pX export signal is conserved in non-human hepatoviruses from a wide range of mammalian species, and functional in pX sequences from bat hepatoviruses when fused to the nanocage protein, suggesting these viruses are released as quasi-enveloped virions. Quantitative proteomics identified multiple ESCRT-related proteins associating with EPN-pX, including ALG2-interacting protein X (ALIX), and its paralog, tyrosine-protein phosphatase non-receptor type 23 (HD-PTP), a second Bro1 domain protein linked to sorting of ubiquitylated cargo into multivesicular endosomes. RNAi-mediated depletion of either Bro1 domain protein impeded eHAV release. Super-resolution fluorescence microscopy demonstrated colocalization of viral capsids with endogenous ALIX and HD-PTP. Co-immunoprecipitation assays using biotin-tagged peptides and recombinant proteins revealed pX interacts directly through the export signal with N-terminal Bro1 domains of both HD-PTP and ALIX. Our study identifies an exceptionally potent viral export signal mediating extracellular release of virus-sized protein assemblies and shows release requires non-redundant activities of both HD-PTP and ALIX.

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The open architecture of HD-PTP phosphatase provides new insights into the mechanism of regulation of ESCRT function.

Publications

The open architecture of HD-PTP phosphatase provides new insights into the mechanism of regulation of ESCRT function.

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