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Vac14 controls PtdIns(3,5)P(2) synthesis and Fab1-dependent protein trafficking to the multivesicular body
Stephen K Dove1, Robert K McEwen, Andrew Mayes
1School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK. s.k.dove@bham.ac.uk
Background:
The PtdIns3P 5-kinase Fab1 makes PtdIns(3,5)P(2), a phosphoinositide essential for retrograde trafficking between the vacuole/lysosome and the late endosome and also for trafficking of some proteins into the vacuole via multivesicular bodies (MVB). No regulators of Fab1 were identified until recently.
Results:
Visual screening of the Eurofan II panel of S. cerevisiae deletion mutants identified YLR386w as a novel regulator of vacuolar function. Others recently identified this ORF as encoding the vacuolar inheritance gene VAC14. Like fab1 mutants, yeast lacking Vac14 have enlarged vacuoles that do not acidify correctly. FAB1 overexpression corrects these defects. vac14Delta cells make very little PtdIns(3,5)P(2), and hyperosmotic shock does not stimulate PtdIns(3,5)P(2) synthesis in the normal manner, implicating Vac14 in Fab1 regulation. We also show that, like fab1Delta mutants, vac14Delta cells fail to sort GFP-Phm5 to the MVB and thence to the vacuole: irreversible ubiquitination of GFP-Phm5 overcomes this defect. In the BY4742 genetic background, loss of Vac14 causes much more penetrant effects on phosphoinositide metabolism and vacuolar trafficking than does loss of Vac7, another regulator of Fab1. Vac14 contains motifs suggestive of a role in protein trafficking and interacts with several proteins involved in clathrin-mediated membrane sorting and phosphoinositide metabolism.
Conclusions:
Vac14 and Vac7 are both upstream activators of Fab1-catalysed PtdIns(3,5)P(2) synthesis, with Vac14 the dominant contributor to the hierarchy of control. Vac14 is essential for the regulated synthesis of PtdIns(3,5)P(2), for control of trafficking of some proteins to the vacuole lumen via the MVB, and for maintenance of vacuole size and acidity.
Insights
Vac14 is a novel regulator of vacuolar function, essential for PtdIns(3,5)P(2) synthesis and protein trafficking. Loss of Vac14 disrupts vacuole size and acidity, impacting cellular processes.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Phosphatidylinositol 3-phosphate 5-kinase (PtdIns3P 5-kinase) Fab1 produces PtdIns(3,5)P(2), crucial for retrograde trafficking and protein transport to the vacuole.
- Previously, no regulators of Fab1 were identified.
Purpose of the Study:
- To identify novel regulators of Fab1 and vacuolar function.
- To elucidate the role of the novel gene YLR386w (Vac14) in PtdIns(3,5)P(2) synthesis and vacuolar trafficking.
Main Methods:
- Visual screening of S. cerevisiae deletion mutants (Eurofan II panel).
- Analysis of vacuolar morphology, acidity, and phosphoinositide levels in wild-type and mutant yeast strains.
- Tracking of fluorescently tagged proteins (GFP-Phm5) to assess trafficking pathways.
Main Results:
- YLR386w, identified as VAC14, is a novel regulator of vacuolar function.
- Vac14 is essential for PtdIns(3,5)P(2) synthesis, vacuolar acidification, and protein trafficking via multivesicular bodies (MVBs).
- Vac14 acts upstream of Fab1, with Vac14 loss causing more severe defects than Vac7 loss.
Conclusions:
- Vac14 is a dominant upstream activator of Fab1-catalyzed PtdIns(3,5)P(2) synthesis.
- Vac14 is critical for regulated PtdIns(3,5)P(2) synthesis, protein trafficking to the vacuole lumen, and maintaining vacuole homeostasis.
- Vac14 plays a key role in cellular signaling and membrane trafficking pathways.