The small GTPase KlRho5 responds to oxidative stress and affects cytokinesis

Abstract

Rho5 is the yeast homolog of the human small GTPase Rac1. We characterized the genes encoding Rho5 and the subunits of its dimeric activating guanine-nucleotide-exchange factor (GEF), Dck1 and Lmo1, in the yeast Kluyveromyces lactis. Rapid translocation of the three GFP-tagged components to mitochondria upon oxidative stress and carbon starvation indicate a similar function of KlRho5 in energy metabolism and mitochondrial dynamics as described for its Saccharomyces cerevisiae homolog. Accordingly, Klrho5 deletion mutants are hyper-resistant towards hydrogen peroxide. Moreover, synthetic lethalities of rho5 deletions with key components in nutrient sensing, such as sch9 and gpr1, are not conserved in K. lactis. Instead, Klrho5 deletion mutants display morphological defects with strengthened lateral cell walls and protruding bud scars. The latter result from aberrant cytokinesis, as observed by following the budding process in vivo and by transmission electron microscopy of the bud neck region. This phenotype can be suppressed by KlCDC42G12V, which encodes a hyper-active variant. Data from live-cell fluorescence microscopy support the notion that KlRho5 interferes with the actin moiety of the contractile actomyosin ring, with consequences different from those previously reported for mutants lacking myosin.

Genetic and Physiological Characterization of Fructose-1,6-Bisphosphate Aldolase and Glyceraldehyde-3-Phosphate Dehydrogenase in the Crabtree-Negative Yeast Kluyveromyces lactis

“…Fluorescent marker genes were fused in frame to the 3′-end of the respective coding sequences by eliminating the translational stop codon of the target gene. The fusion cassettes were equipped with a selectable marker, i.e., kanMX for GFP (pJJH1619) and SkHIS3 for mCherry (pJJH1525), with the template vectors described in [ 66 ]. Strains with different fluorophores were combined as required for colocalizations by crossing and tetrad analyses.…”

The Intracellular Distribution of the Small GTPase Rho5 and Its Dimeric Guanidine Nucleotide Exchange Factor Dck1/Lmo1 Determine Their Function in Oxidative Stress Response

“…For selection of in vivo recombinants, either the kanMX or the SkHIS3 cassette from the Longtine collection [ 43 ], SpHIS5 from pUG27, or KlLEU2 from pUG73 were employed [ 44 ]. GFP tags were amplified from pJJH1619 (GFP-kanMX) or pJJH1620 (GFP-SkHIS3); mCherry tags from pJJH1524 (mCherry-SkHIS3), described in [ 23 ]. As a mitochondrial marker for fluorescence microscopy, either a genomic IDP1-mCherry fusion [ 22 ] was introduced by crossing with the appropriate strains and tetrad analysis, or plasmid pJJH1408 [ 17 ] was employed, which encodes a fusion of the Cox4-mitochondrial signal sequence with mCherry.…”

“…Translocation of homologs of Rho5 and the two GEF subunits to mitochondria was also observed under oxidative and nutrient stress in the more respiratory yeast K. lactis , in which Klrho5 deletions displayed pronounced morphological defects, in contrast to S. cerevisiae [ 23 ]. This is reminiscent of its human homolog Rac1, which, amongst a variety of cellular processes, regulates the dynamics of the actin cytoskeleton [ 24 ].…”

“…Malfunctions of Rac1 have been associated with serious diseases, including cancer and diabetes [ 27 , 28 , 29 , 30 ]. Consequently, yeast expression systems may provide excellent tools to study the molecular functions of the associated RAC1 alleles [ 23 ].…”