The Yeast Phosphatidylinositol Kinase Homolog TOR2 Activates RHO1 and RHO2 via the Exchange Factor ROM2

Abstract

The Saccharomyces cerevisiae phosphatidylinositol kinase homolog TOR2 is required for organization of the actin cytoskeleton. Overexpression of RHO1 or RHO2, encoding Rho-like GTPases, or ROM2, encoding a GDP/GTP exchange factor for RHO1 and RHO2, suppresses a tor2 mutation. Deletion of SAC7, a gene originally identified as a suppressor of an actin mutation, also suppresses a tor2 mutation. SAC7 is a novel GTPase-activating protein for RHO1. ROM2 exchange activity is reduced in a tor2 mutant, and overexpression of ROM2 lacking its PH domain can no longer suppress a tor2 mutation. Thus, TOR2 signals to the actin cytoskeleton through a GTPase switch composed of RHO1, RHO2, ROM2, and SAC7. TOR2 activates this switch via ROM2, possibly via the ROM2 PH domain.

Differential regulation of cell wall biogenesis during growth and development in yeast

“…while SAC7 and BEM2, encoding GTPase-activating proteins (GAPs), decrease Rho1p activity (Schmidt et al, 1997) (see Fig. 3e).…”

The role of ATM in DNA damage responses and cancer

“…ATM is not the only family member associated with cytoplasmic vesicles. TOR2 has been localized to vacuoles in budding yeast and also plays an essential role in the organization of actin cytoskeleton during the cell cycle (Cardenas and Heitman, 1995;Schmidt et al, 1996Schmidt et al, , 1997). …”

Spb4p, an essential putative RNA helicase, is required for a late step in the assembly of 60S ribosomal subunits in Saccharomyces cerevisiae

“…The SPB4 gene was PCR amplified (Vent Polymerase, New England Biolabs) from YCplac111-SPB4 using the reverse primer (59-AAC AGC TAT GAC CAT G-39) and a oligonucleotide introducing the restriction site Sal I (59-GCA GAA TTC GTC GAC TCA AAG TCA TTG GAA TGG GA-39; the Sal I site is underlined, and the SPB4 ORF homology, starting with the second codon, is in bold)+ The PCR product was cut with Sal I and cloned into the Sal I-restricted YCplac111-based plasmid pAS24 (Schmidt et al+, 1997)+ Correct orientation of the Sal I-fragment was verified by restriction analysis+ The resulting construct, pAS24-SPB4, contains a GAL1-10 promoter, a start codon followed by a double HA-tag, and the SPB4 ORF and its 39 contiguous region+ This plasmid was transformed into the strain JDY8-1A YCp50-SPB4+ The subsequent counter-selection of the URA3 SPB4 harboring plasmid on 5-FOA plates containing galactose resulted in the strain JDY8-1A pAS24-SPB4+ The plasmid pAS24-SPB4 complemented the spb4 null strain to the wild-type extent on medium containing galactose (YPGal) at all the tested temperatures (18, 30, and 37 8C)+ We also refer to this strain as the GAL::SPB4 strain or, if grown in medium containing glucose (YPD), as the Spb4p-depleted strain+ For in vivo depletion of Spb4p, JDY8-1A pAS24-SPB4 was grown in liquid YPGal until mid-exponential phase+ Cells were harvested, washed, and used to inoculate YPD cultures+ Cell growth was monitored over a period of 36 h, during which the cultures were regularly diluted into fresh YPD medium to maintain exponential growth+ As a control, JDY8-1A YCplac111-SPB4 was used+ Samples for western blot analyses, polysome analyses, and RNA extraction were taken at different times+…”