Characterization of temperature-sensitive Schizosaccharomyces pombe mutants in the septation initiation network Spg1 GTPase

Abstract

The Schizosaccharomyces pombe GTPase, Spg1 , activates the septation initiation network (SIN) protein kinase cascade to trigger septation. In the absence of functional Spg1 , cells fail cytokinesis and become multinucleate. In this study, we characterize a set of temperature-sensitive spg1 alleles isolated in the 1990s. We identify the mutations within each new and previously characterized allele, characterize the extent of relative growth defects, and assess their interaction with other SIN alleles.

Figure 1. Characterization of spg1 mutant alleles

(A) Schematics of the GTPase Spg1, drawn to scale. The mutations encoded by the listed temperature sensitive alleles are labelled in magenta. (B) The indicated strains were all grown in liquid YE at 25°C until they reached mid-log phase. Then, 10-fold serial dilutions were made and 2.5 µL of each was spotted on YE agar plates and incubated at the indicated temperatures for 2-5 days prior to imaging. (C) The indicated strains were grown at 25°C and shifted to 36°C for 4 hours. Samples were collected at both temperatures and cells were fixed and stained with DAPI and methyl blue. Scale bar, 5 µm. (D) spg1-C1 ura4-D18 cells were transformed with plasmids expressing empty vector (pUR19), pUR19- spg1, or pUR19- cdc7 . Transformants were then streaked to minimal media plates lacking uracil and incubated at 25˚C or 36˚C for 4 days. (E) Ribbon diagram of a structural model of S. pombe Spg1 using AlphaFold2 (Jumper et al., 2021; Varadi et al., 2022) with positions of mutations indicated in magenta.