PXD079187-1

PXD079187 is an original dataset announced via ProteomeXchange.

Title	A GABARAP−PtdIns3K-C1 positive feedback loop at the heart of the phagophore nucleation
Description	Macroautophagy/autophagy is a cellular process enabling degradation of intracellular components during starvation. In mammalian cells, autophagosomes can reach diameters of over 1000 nm within 30 min after triggering starvation, but how such substantial amounts of membranes can be synthesized within a brief time remains elusive. A protein complex central to the phagophore initiation is the lipid kinase PIK3C3-Complex 1 (PtdIns3K-C1), which produces phosphatidylinositol-3-phosphate (PtdIns3P). PtdIns3P recruits a variety of downstream proteins, among which is PtdIns3P-binding WIPI2 that facilitates lipidation of mammalian ATG8 (mATG8) family proteins on phagophores. Here we show that upon acute inhibition of mATG8 lipidation in cells, there is no further accumulation of WIPI2, suggesting a feedback loop between mATG8s and PtdIns3P production. The role of PtdIns3K-C1 in this feedback was demonstrated by in vitro experiments where recombinant membrane-coupled mATG8s bind to and potently activate PtdIns3K-C1, with GABARAP being the most potent activator among all mATG8s. By a combination of cryo-electron microscopy, structural mass spectrometry, activity assays and mutagenesis, we show that GABARAP binds two sites in PtdIns3K-C1, with one site showing an atypical bipartite interaction with the mATG8. We also confirm both sites are essential for GABARAP to activate PtdIns3K-C1. We propose that once GABARAP is indirectly recruited by PtdIns3P generated by basal activity of PtdIns3K-C1, a positive feedback loop is formed where PtdIns3K-C1 interacts with GABARAP and becomes activated to produce more PtdIns3P, thereby further stimulating GABARAP lipidation. This mechanism would be central for autophagosome biogenesis, where enlarged membranes need to be synthesized within a brief period.
HostingRepository	PRIDE
AnnounceDate	2026-07-02
AnnouncementXML	Submission_2026-07-02_05:50:32.804.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Maxime Bourguet
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: NEWT:9606;
ModificationList	No PTMs are included in the dataset
Instrument	Orbitrap Eclipse

Revision	Datetime	Status	ChangeLog Entry
0	2026-06-02 12:24:15	ID requested
⏵ 1	2026-07-02 05:50:33	announced

Dataset with its publication pending

submitter keyword: structural characterization, PI3K, XL-MS, Cryo-EM, HDX-MS,Autophagy

Roger Lee Williams
contact affiliation	MRC - Laboratory of Molecular Biology
contact email	rlw@mrc-lmb.cam.ac.uk
lab head
Maxime Bourguet
contact affiliation	MRC Laboratory of Molecular Biology
contact email	mbourguet@mrc-lmb.cam.ac.uk
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD079187

PRIDE project URI

• PRIDE
  1. PXD079187
     1. Label: PRIDE project
     2. Name: A GABARAP−PtdIns3K-C1 positive feedback loop at the heart of the phagophore nucleation