Bimolecular fluorescence complementation analysis system for <i>in vivo</i> detection of protein–protein interaction in <i>Saccharomyces cerevisiae</i>

Sung, Min Kyung; Huh, Won-Ki

doi:10.1002/yea.1504

Cited by 176 publications

(202 citation statements)

References 32 publications

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“…Briefly, in the BiFC assay, the Venus yellow fluorescent protein (vYFP) is split into 2 fragments, VN (corresponding to the N terminus of vYFP) and VC (the C terminus of vYFP). 48,49 We fused VN to Atg9 and VC to either Atg23 or Atg27 on the genome. Fluorescence from these 2 chimeras can only be detected when the 2 proteins interact and bring the 2 fragments of vYFP proximal to each other.…”

Section: Phosphorylation Of S122 Regulates Atg9 Through Its Interactimentioning

confidence: 99%

Phosphorylation of Atg9 regulates movement to the phagophore assembly site and the rate of autophagosome formation

Feng¹,

Backues²,

Baba³

et al. 2016

Autophagy

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Macroautophagy is primarily a degradative process that cells use to break down their own components to recycle macromolecules and provide energy under stress conditions, and defects in macroautophagy lead to a wide range of diseases. Atg9, conserved from yeast to mammals, is the only identified transmembrane protein in the yeast core macroautophagy machinery required for formation of the sequestering compartment termed the autophagosome. This protein undergoes dynamic movement between the phagophore assembly site (PAS), where the autophagosome precursor is nucleated, and peripheral sites that may provide donor membrane for expansion of the phagophore. Atg9 is a phosphoprotein that is regulated by the Atg1 kinase. We used stable isotope labeling by amino acids in cell culture (SILAC) to identify phosphorylation sites on this protein and identified an Atg1-independent phosphorylation site at serine 122. A nonphosphorylatable Atg9 mutant showed decreased autophagy activity, whereas the phosphomimetic mutant enhanced activity. Electron microscopy analysis suggests that the different levels of autophagy activity reflect differences in autophagosome formation, correlating with the delivery of Atg9 to the PAS. Finally, this phosphorylation regulates Atg9 interaction with Atg23 and Atg27.

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Section: Phosphorylation Of S122 Regulates Atg9 Through Its Interactimentioning

confidence: 99%

Phosphorylation of Atg9 regulates movement to the phagophore assembly site and the rate of autophagosome formation

Feng¹,

Backues²,

Baba³

et al. 2016

Autophagy

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show abstract

“…Briefly, in the BiFC assay, the Venus yellow fluorescent protein (vYFP) is split into 2 fragments, VN (N terminus of vYFP) and VC (C terminus of vYFP). 19 We fused VN to Atg11 on the genome and transformed cells with a plasmid containing Dnm1-VC. Fluorescence from these 2 chimeras can only be detected when the 2 proteins interact and bring the 2 fragments of vYFP proximal to each other.…”

Section: Atg11 Interacts With Dnm1 and Fis1 On The Degrading Peroxisomesmentioning

confidence: 99%

The progression of peroxisomal degradation through autophagy requires peroxisomal division

et al. 2014

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“…Therefore, we decided to reexamine which component of the Atg17-Atg31-Atg29 complex was the direct binding partner of Atg11. To carry out this analysis, we took advantage of the bimolecular fluorescence complementation assay (35). In this system, the Venus YFP is split into two fragments, corresponding to the N terminus (VN) and C terminus (VC).…”

Section: Significancementioning

confidence: 99%

Atg29 phosphorylation regulates coordination of the Atg17-Atg31-Atg29 complex with the Atg11 scaffold during autophagy initiation

Mao

Chew

Inoue-Aono

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Macroautophagy (hereafter autophagy) functions in the nonselective clearance of cytoplasm. This process participates in many aspects of cell physiology, and is conserved in all eukaryotes. Autophagy begins with the organization of the phagophore assembly site (PAS), where most of the AuTophaGy-related (Atg) proteins are at least transiently localized. Autophagy occurs at a basal level and can be induced by various types of stress; the process must be tightly regulated because insufficient or excessive autophagy can be deleterious. A complex composed of Atg17-Atg31-Atg29 is vital for PAS organization and autophagy induction, implying a significant role in autophagy regulation. In this study, we demonstrate that Atg29 is a phosphorylated protein and that this modification is critical to its function; alanine substitution at the phosphorylation sites blocks its interaction with the scaffold protein Atg11 and its ability to facilitate assembly of the PAS. Atg29 has the characteristics of an intrinsically disordered protein, suggesting that it undergoes dynamic conformational changes on interaction with a binding partner(s). Finally, single-particle electron microscopy analysis of the Atg17-Atg31-Atg29 complex reveals an elongated structure with Atg29 located at the opposing ends.lysosome | organelle biogenesis | vacuole | yeast

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Bimolecular fluorescence complementation analysis system for in vivo detection of protein–protein interaction in Saccharomyces cerevisiae

Cited by 176 publications

References 32 publications

Phosphorylation of Atg9 regulates movement to the phagophore assembly site and the rate of autophagosome formation

Phosphorylation of Atg9 regulates movement to the phagophore assembly site and the rate of autophagosome formation

The progression of peroxisomal degradation through autophagy requires peroxisomal division

Atg29 phosphorylation regulates coordination of the Atg17-Atg31-Atg29 complex with the Atg11 scaffold during autophagy initiation

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