Katanaev, V. L. | bioRxiv Science

bioRxiv2020

Local and substrate-specific S-palmitoylation determines subcellular localization of Gαo

Peripheral membrane proteins (PMPs) associate with cellular membranes through post-translational modifications like S-palmitoylation. The Golgi apparatus is generally viewed as the transitory station where palmitoyl acyltransferases (PATs) modify PMPs, which are then transported to their ultimate destinations such as the plasma membrane (PM). However, little substrate specificity among the many PATs has been determined. Here we describe the inherent partitioning of Go - -subunit of heterotrimeric Go proteins - to PM and Golgi, independent from Golgi-to-PM transport. A minimal code within Go N-terminus governs its compartmentalization and re-coding produces G protein versions with shifted localization. We establish the S-palmitoylation at the outer nuclear membrane assay ("SwissKASH") to probe substrate specificity of PATs in intact cells. With this assay, we show that PATs localizing to different membrane compartments display remarkable substrate selectivity, which is the basis for PMP compartmentalization. Our findings uncover a mechanism governing protein localization and establish the basis for innovative drug discovery.

cell biology↗

bioRxiv2020

Humanization of Drosophila Gαo to model GNAO1 paediatric encephalopathies

Several hundred genes have been identified to contribute to epilepsy - the disease affecting 65 million people worldwide. One of these genes is GNAO1 encoding Go, the major neuronal -subunit of heterotrimeric G proteins. An avalanche of dominant de novo mutations in GNAO1 have been recently described in paediatric epileptic patients, suffering in addition to epilepsy from motor dysfunction and developmental delay. Although occurring in amino acids conserved from humans to Drosophila, these mutations and their functional consequences have only poorly been analysed at the biochemical or neuronal levels. Adequate animal models to study molecular aetiology of GNAO1 encephalopathies have also so far been lacking. As the first step towards modelling the disease in Drosophila, we here describe humanization of the Go locus in the fruit fly. A two-step CRISPR/Cas9-mediated replacement was conducted, first substituting the coding exons 2-3 of Go with respective human GNAO1 sequences. At the next step, the remaining exons 4-7 were similarly replaced, keeping intact the gene Cyp49a1 embedded in-between, as well as the non-coding exon 1 and the surrounding regulatory sequences. The resulting flies, homozygous for the humanized GNAO1 loci, are viable and fertile without any visible phenotypes; their body weight and longevity are also normal. Human Go-specific antibodies confirm the endogenous-level expression of the humanized Go, which fully replaces the Drosophila functions. The genetic model we established will make it easy to incorporate encephalopathic GNAO1 mutations and will permit intensive investigations into the molecular aetiology of the human disease through the powerful toolkit of Drosophila genetics.

genetics↗