GAPR-1 Interferes with Condensate Formation of Beclin 1 in Saccharomyces cerevisiae. Issue 4 (28th February 2023)

• Record Type: Journal Article
• Authors: Sirati, Nafiseh; Shen, Ziying; Olrichs, Nick K.; Popova, Blagovesta; Verhoek, Iris C.; Lagerwaard, Ilse M.; Braus, Gerhard H.; Kaloyanova, Dora V.; Helms, J.Bernd
• Is Part Of: Journal of molecular biology. Volume 435:Issue 4(2023)
• Publication Date: 2023-02-28
• Subjects: GLIPR2 -- CAP superfamily -- amyloid-like proteins -- protein–protein interactions (PPIs) -- autophagy; GAPR-1 Golgi-Associated plant Pathogenesis Related protein 1 -- PI3K Class III Phosphatidylinositol 3-Kinase -- Vps34 Vacuolar protein sorting 34 -- Bcl-2 B cell lymphoma 2 -- CAP Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 proteins -- IP6 Inositol hexakisphosphate -- S. cerevisiae Saccharomyces cerevisiae -- Aβ Amyloid β -- Htt Huntingtin -- GFP Green fluorescent protein -- SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis -- ADH1 Alcohol dehydrogenase I -- BiFC Bimolecular Complementation -- ThT Thioflavin T -- 1, 6-HD 1, 6-Hexanediol -- YFP Yellow fluorescent protein -- PPIs Protein–Protein Interactions -- ECD Evolutionarily conserved domain -- GAPDH Glyceraldehyde 3-phosphate dehydrogenase -- SC Synthetic Complete; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2022.167935
• Languages: English
• ISSNs: 0022-2836

Abstract

Graphical abstract: Highlights: hBeclin 1 expression in yeast results in the formation of protein condensates. hBeclin 1 condensate formation is inhibited by hGAPR-1. Previously described PPIs affect hBeclin 1 and hGAPR-1 condensate formation. We propose a novel assay system to study PPIs between amyloidogenic proteins. Abstract: Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1) acts as a negative regulator of autophagy by interacting with Beclin 1 at Golgi membranes in mammalian cells. The molecular mechanism of this interaction is largely unknown. We recently showed that human GAPR-1 (hGAPR-1) has amyloidogenic properties resulting in the formation of protein condensates upon overexpression in Saccharomyces cerevisiae. Here we show that human Beclin 1 (hBeclin 1) has several predicted amyloidogenic regions and that overexpression of hBeclin 1-mCherry in yeast also results in the formation of fluorescent protein condensates. Surprisingly, co-expression of hGAPR-1-GFP and hBeclin 1-mCherry results in a strong reduction of hBeclin 1 condensates. Mutations of the known interaction site on the hGAPR-1 and hBeclin 1 surface abolished the effect on condensate formation during co-expression without affecting the condensate formation properties of the individual proteins. Similarly, a hBeclin 1-derived B18 peptide that is known to bind hGAPR-1 and to interfere with the interaction between hGAPR-1 and hBeclin 1, abolished the reduction of hBeclin 1 condensates by co-expression of hGAPR-1. These results indicate that the same type of protein–protein interactions interfere with condensate formation during co-expression of hGAPR-1 and hBeclin 1 as previously described for their interaction at Golgi membranes. The amyloidogenic properties of the B18 peptide were, however, important for the interaction with hGAPR-1, as mutant peptides with reduced amyloidogenic properties also showed reduced interaction with hGAPR-1 and reduced interference with hGAPR-1/hBeclin 1 condensate formation. We propose that amyloidogenic interactions take place between hGAPR-1 and hBeclin 1 prior to condensate formation.