Knockout of RSN1, TVP18 or CSC1‐2 causes perturbation of Golgi cisternae in Pichia pastoris

• DOI: 10.1111/tra.12773
• Publication Date: 2020-12-02T10:11:07Z
• Authors: Rochelle Aw; Charlot De Wachter; Bram Laukens; Riet De Rycke; Michiel De Bruyne; David Bell; Nico Callewaert; Karen M. Polizzi
• Keywords: SELECTION; Pichia pastoris/Komagataella phaffi; 0301 basic medicine; 570; glycosylation; PROTEINS; Golgi Apparatus; ORGANIZATION; Saccharomyces cerevisiae; 0601 Biochemistry and Cell Biology; 03 medical and health sciences; Pichia pastoris; 1108 Medical Microbiology; Golgi; Animals; TRAFFICKING; STACKING; Science & Technology; COMPLEX; Secretory Pathway; IDENTIFICATION; Komagataella phaffi; GLYCOSYLATION; Proteins; Cell Biology; stacked cisternae; secretory pathway; HOMOLOG; CIS-GOLGI; Saccharomycetales; Life Sciences & Biomedicine; Developmental Biology

AbstractThe structural organization of the Golgi stacks in mammalian cells is intrinsically linked to function, including glycosylation, but the role of morphology is less clear in lower eukaryotes. Here we investigated the link between the structural organization of the Golgi and secretory pathway function using Pichia pastoris as a model system. To unstack the Golgi cisternae, we disrupted 18 genes encoding proteins in the secretory pathway without loss of viability. Using biosensors, confocal microscopy and transmission electron microscopy we identified three strains with irreversible perturbations in the stacking of the Golgi cisternae, all of which had disruption in genes that encode proteins with annotated function as or homology to calcium/calcium permeable ion channels. Despite this, no variation in the secretory pathway for ER size, whole cell glycomics or recombinant protein glycans was observed. Our investigations showed the robust nature of the secretory pathway in P. pastoris and suggest that Ca2+ concentration, homeostasis or signalling may play a significant role for Golgi stacking in this organism and should be investigated in other organisms.