Genome-wide analysis of intracellular pH reveals quantitative control of cell division rate by pHc in Saccharomyces cerevisiae.

BACKGROUND: Because protonation affects the properties of almost all molecules in cells, cytosolic pH (pH(c)) is usually assumed to be constant. In the model organism yeast, however, pH(c )changes in response to the presence of nutrients and varies during growth. Since small changes in pH(c )can lead to major changes in metabolism, signal transduction, and phenotype, we decided to analyze pH(c )control. RESULTS: Introducing a pH-sensitive reporter protein into the yeast deletion collection allowed quantitative genome-wide analysis of pH(c )in live, growing yeast cultures. pH(c )is robust towards gene deletion; no single gene mutation led to a pH(c )of more than 0.3 units lower than that of wild type. Correct pH(c )control required not only vacuolar proton pumps, but also strongly relied on mitochondrial function. Additionally, we identified a striking relationship between pH(c )and growth rate. Careful dissection of cause and consequence revealed that pH(c )quantitatively controls growth rate. Detailed analysis of the genetic basis of this control revealed that the adequate signaling of pH(c )depended on inositol polyphosphates, a set of relatively unknown signaling molecules with exquisitely pH sensitive properties. CONCLUSIONS: While pH(c )is a very dynamic parameter in the normal life of yeast, genetically it is a tightly controlled cellular parameter. The coupling of pH(c )to growth rate is even more robust to genetic alteration. Changes in pH(c )control cell division rate in yeast, possibly as a signal. Such a signaling role of pH(c )is probable, and may be central in development and tumorigenesis

Hansenula polymorpha Swi1p and Snf2p are essential for methanol utilisation

We have cloned the Hansenula polymorpha SWI1 and SNF2 genes by functional complementation of mutants that are defective in methanol utilisation. These genes encode proteins similar to Saccharomyces cerevisiae Swi1p and Snf2p, which are subunits of the SWI/SNF complex. This complex belongs to the family of nucleosome-remodeling complexes that play a role in transcriptional control of gene expression. Analysis of the phenotypes of constructed H. polymorpha SWI1 and SNF2 disruption strains indicated that these genes are not necessary for growth of cells on glucose, sucrose, or various organic nitrogen sources which involve the activity of peroxisomal oxidases. Both disruption strains showed a moderate growth defect on glycerol and ethanol, but were fully blocked in methanol utilisation. In methanol-induced cells of both disruption strains, two peroxisomal enzymes involved in methanol metabolism, alcohol oxidase and dihydroxyacetone synthase, were hardly detectable, whereas in wild-type cells these proteins were present at very high levels. We show that the reduction in alcohol oxidase protein levels in H. polymorpha SWI1 and SNF2 disruption strains is due to strongly reduced expression of the alcohol oxidase gene. The level of Pex5p, the receptor involved in import of alcohol oxidase and dihydroxyacetone synthase into peroxisomes, was also reduced in both disruption strains compared to that in wild-type cells.

Hsp110 is required for spindle length control.

Systematic affinity purification combined with mass spectrometry analysis of N- and C-tagged cytoplasmic Hsp70/Hsp110 chaperones was used to identify new roles of Hsp70/Hsp110 in the cell. This allowed the mapping of a chaperone-protein network consisting of 1,227 unique interactions between the 9 chaperones and 473 proteins and highlighted roles for Hsp70/Hsp110 in 14 broad biological processes. Using this information, we uncovered an essential role for Hsp110 in spindle assembly and, more specifically, in modulating the activity of the widely conserved kinesin-5 motor Cin8. The role of Hsp110 Sse1 as a nucleotide exchange factor for the Hsp70 chaperones Ssa1/Ssa2 was found to be required for maintaining the proper distribution of kinesin-5 motors within the spindle, which was subsequently required for bipolar spindle assembly in S phase. These data suggest a model whereby the Hsp70-Hsp110 chaperone complex antagonizes Cin8 plus-end motility and prevents premature spindle elongation in S phase