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The carboxy terminus of yeast Atg13 binds phospholipid membrane via motifs that overlap with the Vac8-interacting domain.

ABSTRACT: Macroautophagy/autophagy is a conserved catabolic recycling pathway involving the sequestration of cytoplasmic components within double-membrane vesicles termed autophagosomes. The autophagy-related (Atg) protein Atg13 is a key member of the autophagy initiation complex. The Atg13 C terminus is an intrinsically disordered region (IDR) harboring a binding site for the vacuolar membrane protein Vac8. Recent reports suggest Atg13 acts as a hub to assemble the initiation complex, and also participates in membrane recognition. Here we show that the Atg13 C terminus directly binds to lipid membranes via electrostatic interactions between positively charged residues in Atg13 and negatively charged phospholipids as well as a hydrophobic insertion of a Phe residue. We identified 2 sets of residues in the Atg13 IDR that affect its phospholipid-binding properties; these residues overlap with the Vac8-binding domain of Atg13. Our data indicate that Atg13 binding to phospholipids and Vac8 is mutually exclusive, and both are required for efficient autophagy.

Abbreviations

Atg: autophagy-related; CD: circular dichroism; Cvt: cytoplasm-to-vacuole targeting; IDR: intrinsically disordered region; ITC: isothermal calorimetry; MIM: MIT-interacting motif; MKO: multiple-knockout; PAS: phagophore assembly site; PC: phosphatidylcholine; PS: phosphatidylserine; PtdIns: phosphatidylinositol; PtdIns3P: phosphatidylinositol-3-phosphate.

SUBMITTER: Gatica D

PROVIDER: S-EPMC7469566 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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The carboxy terminus of yeast Atg13 binds phospholipid membrane via motifs that overlap with the Vac8-interacting domain.

Gatica Damián D Damasio Alejandro A Pascual Clarence C Klionsky Daniel J DJ Ragusa Michael J MJ Popelka Hana H

Autophagy 20190802 6

Macroautophagy/autophagy is a conserved catabolic recycling pathway involving the sequestration of cytoplasmic components within double-membrane vesicles termed autophagosomes. The autophagy-related (Atg) protein Atg13 is a key member of the autophagy initiation complex. The Atg13 C terminus is an intrinsically disordered region (IDR) harboring a binding site for the vacuolar membrane protein Vac8. Recent reports suggest Atg13 acts as a hub to assemble the initiation complex, and also participat ...[more]

PMID: 31352862

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Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) is a human oncogenic virus and the causative agent of Kaposi's sarcoma, multicentric Castleman's disease, and primary effusion lymphoma. During lytic reactivation, there is a temporal cascade of viral gene expression that results in the production of new virions. One of the viral factors that is expressed during reactivation is open reading frame 59 (ORF59), the viral DNA polymerase processivity factor. ORF59 plays an essential role for DNA synthesis and is required for the nuclear localization of the viral DNA polymerase (ORF9) to the origin of lytic replication (oriLyt). In addition to its functions in viral DNA synthesis, ORF59 has been shown to interact with chromatin complexes, including histones and cellular methyltransferases. In this study, a series of KSHV BACmids containing 50-amino acid (aa) deletions within ORF59 were generated to determine the interaction domains between ORF59 and histones, as well as to assess the effects on replication fitness as a result of these interactions. These studies show that in the context of infection, ORF59 51 to 100 and 151 to 200 amino acids (aa) are required for interaction with histones, and ORF59 301 to 396 aa are not required for DNA synthesis. Since full-length ORF59 is known to localize to the nucleus, we performed an immunofluorescent assay (IFA) with the ORF59 deletion mutants and showed that all deletions are localized to the nucleus; this includes the ORF59 deletion without the previously identified nuclear localization signal (NLS). These studies further characterize ORF59 and demonstrate its essential role during lytic replication.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus and the causative agent of potentially fatal malignancies. Lytic replication of KSHV is an essential part of the viral life cycle, allowing for virus dissemination within the infected host and shedding to infect naive hosts. Viral DNA synthesis is a critical step in the production of new infectious virions. One of the proteins that is vital to this process is open reading frame 59 (ORF59), the viral encoded polymerase processivity factor. Previous work has demonstrated that the function of ORF59 is closely connected to its association with other viral and cellular factors. The studies presented here extend that work to include the interaction between ORF59 and histones. This interaction offers an additional level of regulation of the chromatinized viral genome, ultimately influencing DNA synthesis and transcription dynamics.

Dataset Information

The carboxy terminus of yeast Atg13 binds phospholipid membrane via motifs that overlap with the Vac8-interacting domain.

Abbreviations

Publications

The carboxy terminus of yeast Atg13 binds phospholipid membrane via motifs that overlap with the Vac8-interacting domain.

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