Quantitative analysis of human ras localization and function in the fission yeast Schizosaccharomyces pombe

Abstract

Ras signalling is central to fundamental and diverse cellular processes. In higher eukaryotes ras signalling is highly complex, involving multiple isoforms, regulatory proteins and effectors. As a consequence, the study of ras activity in mammalian systems presents a number of technical challenges. The model organism Schizosaccharomyces pombe has previously proved a key system for the study of human signalling components and provides an ideal model for the study of ras, as it contains just one ras protein (Ras1p), which is non-essential and controls a number of downstream processes. Here we present data demonstrating the quantitative analysis of three distinct Ras1-related signalling outputs, utilizing the three most abundant human ras isoforms, H-Ras, N-Ras and K-Ras4B, in Sz. pombe. Further, we have characterized the localization of these three human ras isoforms in Sz. pombe, utilizing quantitative image analysis techniques. These data indicate that all three human ras isoforms are functional in fission yeast, displaying differing localization patterns which correlate strongly with function in the regulation of pheromone response and cell shape. These data demonstrate that such yeast strains could provide powerful tools for the investigation of ras biology, and potentially in the development of cancer therapies.

Investigating G protein signalling bias at the glucagon‐like peptide‐1 receptor in yeast

“…HRP activity was detected using enhanced chemiluminescence reagents EZ‐ECL (Geneflow, Lichfield, UK) and visualized using a Syngene G:Box gel documentation system (Syngene, Cambridge, UK) as described in Bond et al . ().…”

“…Western blotting was performed using a GLP-1 receptor polyclonal antibody (Abcam, Cambridge, UK) and a donkey anti-rabbit IgG HRP conjugate (Amersham, International, Little Chalfont, UK) as the secondary antibody. HRP activity was detected using enhanced chemiluminescence reagents EZ-ECL (Geneflow, Lichfield, UK) and visualized using a Syngene G:Box gel documentation system (Syngene, Cambridge, UK) as described in Bond et al (2013).…”

Modulation of Glucagon Receptor Pharmacology by Receptor Activity-modifying Protein-2 (RAMP2)

“…Relative efficacy (log τ) and equilibrium dissociation constants (log K A ) were generated through use of an operational model for partial agonism ( 23 ). Fluorescent image analysis was performed using ImageJ version 1.46b using the QUIMP plugin as described previously ( 17 , 24 ). Statistical differences were analyzed before data were normalized, using a Student's test or one-way analysis of variance (ANOVA) with Bonferroni's or Dunnett's multiple comparisons as appropriate, and p < 0.05 was considered significant.…”

Membrane Thickness as a Key Factor Contributing to the Activation of Osmosensors and Essential Ras Signaling Pathways

“…Several studies have demonstrated that there is a functional interchangeability between the yeast Ras1 and mammalian H-Ras (DeFeo-Jones et al, 1985 ; Bond et al, 2013 ), a finding indicative of the conservation of similar membrane activation mechanisms, possibly including lateral transfer into distinct nanoclusters upon activation. In this regard, it is known that the contribution of amino acids R169 and K170 at the HVR-1 domain of H-Ras to the lateral segregation requires that this region to be correctly spaced relative to the location of the membrane lipidic anchors (Rotblat et al, 2004 ).…”