Dynamic and Reversible Aggregation of the Human CAP Superfamily Member GAPR-1 in Protein Inclusions in Saccharomyces cerevisiae. Issue 19 (17th September 2021)

• Record Type: Journal Article
• Authors: Sirati, Nafiseh; Popova, Blagovesta; Molenaar, Martijn R.; Verhoek, Iris C.; Braus, Gerhard H.; Kaloyanova, Dora V.; Helms, J. Bernd
• Is Part Of: Journal of molecular biology. Volume 433:Issue 19(2021)
• Publication Date: 2021-09-17
• Subjects: GLIPR-2 -- condensates -- amyloids -- zinc -- myristoylation; GAPR-1 Golgi-Associated plant Pathogenesis Related protein 1 -- CAP Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 proteins -- GFP Green fluorescent protein -- Aβ Amyloid β -- TDP-43 TAR DNA-binding protein 43 -- FUS Fused in Sarcoma -- LLPS Liquid-liquid phase separation -- BS3 Bis (sulfosuccinimidyl) suberate -- ThT Thioflavin T -- SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis -- FRAP Fluorescence Recovery After Photobleaching -- GAPDH Glyceraldehyde 3-phosphate dehydrogenase -- SC Synthetic Complete -- WB Western blot; 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.2021.167162
• Languages: English
• ISSNs: 0022-2836

Abstract

Graphical abstract: Highlights: GAPR-1-GFP expression in yeast results in the formation of protein inclusions. Inclusions formed by GAPR-1-GFP are dynamic and reversible structures. N-myristoylation and Zinc-binding regulate GAPR-1 inclusion formation. Similar factors control inclusion formation and amyloid-like aggregation in vitro. Abstract: Many proteins that can assemble into higher order structures termed amyloids can also concentrate into cytoplasmic inclusions via liquid–liquid phase separation. Here, we study the assembly of human Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1), an amyloidogenic protein of the Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 proteins (CAP) protein superfamily, into cytosolic inclusions in Saccharomyces cerevisiae . Overexpression of GAPR-1-GFP results in the formation GAPR-1 oligomers and fluorescent inclusions in yeast cytosol. These cytosolic inclusions are dynamic and reversible organelles that gradually increase during time of overexpression and decrease after promoter shut-off. Inclusion formation is, however, a regulated process that is influenced by factors other than protein expression levels. We identified N-myristoylation of GAPR-1 as an important determinant at early stages of inclusion formation. In addition, mutations in the conserved metal-binding site (His54 and His103) enhanced inclusion formation, suggesting that these residues prevent uncontrolled protein sequestration. In agreement with this, we find that addition of Zn 2+ metal ions enhances inclusion formation. Furthermore, Zn 2+ reduces GAPR-1 protein degradation, which indicates stabilization of GAPR-1 in inclusions. We propose that the properties underlying both the amyloidogenic properties and the reversible sequestration of GAPR-1 into inclusions play a role in the biological function of GAPR-1 and other CAP family members.