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The Third Coiled Coil Domain of Atg11 Is Required for Shaping Mitophagy Initiation Sites.

ABSTRACT: Selective autophagy is the capture of specific cytosolic contents in double-membrane vesicles that subsequently fuse with the vacuole or lysosome, thereby delivering cargo for degradation. Selective autophagy receptors (SARs) mark the cargo for degradation and, in yeast, recruit Atg11, the scaffolding protein for selective autophagy initiation. The mitochondrial protein Atg32 is the yeast SAR that mediates mitophagy, the selective autophagic capture of mitochondria. Atg11-Atg32 interactions concentrate Atg32 into puncta that are thought to represent sites of mitophagy initiation. However, it is unclear how Atg11 concentrates Atg32 to generate mitophagy initiation sites. We show here that the coiled coil 3 (CC3) domain of Atg11 is required for concentrating Atg32 into puncta. We determined the structure of the majority of the CC3, demonstrating that the CC3 forms a parallel homodimer whose dimer interface is formed by a small number of hydrophobic residues. We further show that the CC3 interface is not required for Atg11 dimerization but is required for shaping Atg32 into functional mitophagy initiation sites and for delivery of mitochondria to the vacuole. Our findings suggest that Atg11 self-interactions help concentrate SARs as a necessary precondition for cargo capture.

SUBMITTER: Margolis HK

PROVIDER: S-EPMC7603899 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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The Third Coiled Coil Domain of Atg11 Is Required for Shaping Mitophagy Initiation Sites.

Margolis Hannah K HK Katzenell Sarah S Leary Kelsie A KA Ragusa Michael J MJ

Journal of molecular biology 20200905 21

Selective autophagy is the capture of specific cytosolic contents in double-membrane vesicles that subsequently fuse with the vacuole or lysosome, thereby delivering cargo for degradation. Selective autophagy receptors (SARs) mark the cargo for degradation and, in yeast, recruit Atg11, the scaffolding protein for selective autophagy initiation. The mitochondrial protein Atg32 is the yeast SAR that mediates mitophagy, the selective autophagic capture of mitochondria. Atg11-Atg32 interactions conc ...[more]

PMID: 32896530

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Project description:Two types of viruses are produced during the baculovirus life cycle: budded virus (BV) and occlusion-derived virus (ODV). A particular baculovirus protein, FP25K, is involved in the switch from BV to ODV production. Previously, FP25K from the model alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was shown to traffic ODV envelope proteins. However, FP25K localization and the domains involved are inconclusive. Here we used a quantitative approach to study FP25K subcellular localization during infection using an AcMNPV bacmid virus that produces a functional AcMNPV FP25K-green fluorescent protein (GFP) fusion protein. During cell infection, FP25K-GFP localized primarily to the cytoplasm, particularly amorphous structures, with a small fraction being localized in the nucleus. To investigate the sequences involved in FP25K localization, an alignment of baculovirus FP25K sequences revealed that the N-terminal putative coiled-coil domain is present in all alphabaculoviruses but absent in betabaculoviruses. Structural prediction indicated a strong relatedness of AcMNPV FP25K to long interspersed element 1 (LINE-1) open reading frame 1 protein (ORF1p), which contains an N-terminal coiled-coil domain responsible for cytoplasmic retention. Point mutations and deletions of this domain lead to a change in AcMNPV FP25K localization from cytoplasmic to nuclear. The coiled-coil and C-terminal deletion viruses increased BV production. Furthermore, a betabaculovirus FP25K protein lacking this N-terminal coiled-coil domain localized predominantly to the nucleus and exhibited increased BV production. These data suggest that the acquisition of this N-terminal coiled-coil domain in FP25K is important for the evolution of alphabaculoviruses. Moreover, with the divergence of preocclusion nuclear membrane breakdown in betabaculoviruses and membrane integrity in alphabaculoviruses, this domain represents an alphabaculovirus adaptation for nuclear trafficking of occlusion-associated proteins.ImportanceBaculovirus infection produces two forms of viruses: BV and ODV. Manufacturing of ODV involves trafficking of envelope proteins to the inner nuclear membrane, mediated partly through the FP25K protein. Since FP25K is present in alpha-, beta-, and gammabaculoviruses, it is uncertain if this trafficking function is conserved. In this study, we looked at alpha- and betabaculovirus FP25K trafficking by its localization. Alphabaculovirus FP25K localized primarily to the cytoplasm, whereas betabaculovirus FP25K localized to the nucleus. We found that an N-terminal coiled-coil domain present in all alphabaculovirus FP25K proteins, but absent in betabaculovirus FP25K, was critical for alphabaculovirus FP25K cytoplasmic localization. We believe that this represents an evolutionary process that partly led to the gain of function of this N-terminal coiled-coil domain in alphabaculovirus FP25K to aid in nuclear trafficking of occlusion-associated proteins. Due to betabaculovirus breakdown of the nuclear membrane before occlusion, this function is not needed, and the domain was lost or never acquired.

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Dataset Information

The Third Coiled Coil Domain of Atg11 Is Required for Shaping Mitophagy Initiation Sites.

Publications

The Third Coiled Coil Domain of Atg11 Is Required for Shaping Mitophagy Initiation Sites.

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