Dataset Information

Lgl protein interactome - Lgl reduces endosomal vesicle acidification and Notch signaling by promoting the interaction between Vap33 and the V-ATPase complex

ABSTRACT: To identify Lgl binding proteins, we purified Lgl-containing protein complexes from cultured Drosophila S2 cells, using the single-step streptavidin purification from stable cell lines expressing SBP-tagged Lgl, followed by nanoLC-MS/MS analysis of the interactors. This study revealed the binding of Lgl to Vap33, which we further connected to the function of the vacuolar ATPase and regulation of Notch signaling.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Drosophila Melanogaster (fruit Fly)

TISSUE(S): Cell Culture, Early Embryonic Cell

SUBMITTER: Alexey Veraksa

LAB HEAD: Alexey Veraksa

PROVIDER: PXD009568 | Pride | 2018-06-10

REPOSITORIES: Pride

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Lgl reduces endosomal vesicle acidification and Notch signaling by promoting the interaction between Vap33 and the V-ATPase complex.

Portela Marta M Yang Liu L Paul Sayantanee S Li Xia X Veraksa Alexey A Parsons Linda M LM Richardson Helena E HE

Science signaling 20180605 533

Epithelial cell polarity is linked to the control of tissue growth and tumorigenesis. The tumor suppressor and cell polarity protein lethal-2-giant larvae (Lgl) promotes Hippo signaling and inhibits Notch signaling to restrict tissue growth in <i>Drosophila melanogaster</i> Notch signaling is greater in <i>lgl</i> mutant tissue than in wild-type tissue because of increased acidification of endosomal vesicles, which promotes the proteolytic processing and activation of Notch by γ-secretase. We sh ...[more]

PMID: 29871910

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Project description:Circadian rhythms are daily oscillations in metabolism and physiology and are generated by the circadian clock. In fruit fly Drosophila, the circadian clock is generated by a transcription-translation feedback loop in which the positive arm components Clock and Cycle activate the expression of the Period and Timeless genes of negative arm, as well as other circadian clock-regulated genes. After being retained in the cytoplasm, the Period and Timeless proteins then migrate to the nucleus to inhibit the Clock/Cycle transactivity by protein-protein interactions (PPIs). The endogenous circadian clock is synchronized with the geological (solar) clock via photoreceptors. Drosophila Cryptochrome protein functions as a circadian photoreceptor. In the early morning, exposure of Cryptochrome to light causes a conformational change in it which results in the formation of new PPIs. Light-activated Cryptochrome interacts with the core clock protein Timeless and the E3 ubiquitin ligase-substrate adaptor protein Jetlag, which results in the ubiquitylation of Timeless by Jetlag-E3 ligase complex and then degradation of Timeless within minutes by proteasome system. Rapid degradation of Timeless and then its partner protein Period, because of its instability in the absence of Timeless, relieves the inhibition on the Clock/Cycle transcription factors suddenly. Therefore, Clock/Cycle-driven expression of circadian clock-regulated genes are induced again, which is the restart of the circadian oscillation or the resetting of the clock. Following Timeless degradation, Cryptochrome is also degraded so the photoreceptor mechanism does not start a new resetting signal until all the required factors are re-synthesized in a circadian manner. Light-dependent degradation of Drosophila Cryptochrome can be observed in Drosophila S2 cell line in culture. In this project, we aimed at finding the interactome of Cryptochrome protein in Drosophila S2 cell line under light and in the dark using proximity labeling method. Because of the fast kinetics of Cryptochrome degradation, we chose the enzymes that can saturate in less than one hour. TurboID (TID) and APEX2 enzymes label proteins with biotin in the proximity even though they work with different mechanisms. They were fused to Cryptochrome protein, and proximity labeling was performed in the dark or under light. We have identified novel light-dependent or -independent interactors of Drosophila Cryptochrome and confirmed some of them using classical coimmunoprecipitation technique.

			Action	DRS
	20421.RAW	Raw
	20421peptides.csv	Csv
	20421proteins.csv	Csv
	20422.RAW	Raw
	20422peptides.csv	Csv