Evolution and functional analysis of the intrinsically disordered N-terminal region of the oncoprotein GOLPH3 in vertebrates.

Abstract

GOLPH3 is a peripheral membrane oncoprotein that in mammals is localized in the trans-Golgi network. This protein provides essential functions in vesicle trafficking and Golgi structure. However, elevated levels of GOLPH3 can trigger cell alterations. Moreover, several studies show a high expression of the gene that encodes this protein in several solid tumor types. GOLPH3 structure comprises a globular C-terminal portion of unique folding and a predicted intrinsically disordered region (IDR) located in the N-terminal portion of the protein, for which its function is unknown. It is known that IDRs are present in many proteins that form biomolecular condensates, which are membraneless compartments. Additionally, evolutionary studies in GOLPH3 suggest that this IDR performs a specialized function that remains to be elucidated. This thesis aims to evaluate the function of the IDR of GOLPH3 that we postulate is involved in the formation of biomolecular condensates, and how the amino acid sequence divergence among vertebrate species affects this capability. Through evolutionary analysis, N-terminal of fourth vertebrate species were selected to produce recombinant proteins. To analyze the functional effect, we expressed GOLPH3 variants in human cultured cells and performed phase separation assays with recombinant proteins. We observed that the protein stability of GOLPH3 variants is similar to human wild-type GOLPH3 and the IDR of the selected sequences does not affect GOLPH3 localization in human cells. According to our results, the IDR is necessary for in vitro liquid-liquid phase separation (LLPS) of GOLPH3, suggesting that the N-terminal of GOLPH3 could be important in the formation of the uncharacterized GOLPH3 membraneless compartment found in cultured cells, a feature that could be relevant for its biological function. Unexpectedly, we obtained GOLPH3-full-length variants crystallization, finding that could be used for further structural and functional characterization of the GOLPH3 family of proteins.