SUMMARY During yeast cell division, aggregates of damaged proteins are segregated asymmetrically between the bud and the mother. It is thought that protein aggregates are cleared from the bud via actin cable-based retrograde transport toward the mother, and that Bni1p formin regulates this transport. Here we examined the dynamics of Hsp104-associated protein aggregates by video microscopy, particle tracking and image correlation analysis. We show that protein aggregates undergo random walk without directional bias. Clearance of heat-induced aggregates from the bud does not depend on formin proteins but occurs mostly through dissolution via Hsp104p chaperon. Aggregates formed naturally in aged cells also exhibit random walk but do not dissolve during observation. Although our data does not disagree with a role for actin or cell polarity in aggregate segregation, modeling suggests that their asymmetric inheritance can be a predictable outcome of aggregates' slow diffusion and the geometry of yeast cells.
Cellular aging is known to correlate with the accumulation of many harmful agents1, but can aging also result from deterioration of certain poorly-renewed beneficial components? Here we found that a group of plasma membrane-associated transporters, belonging to the multidrug resistance (MDR) protein families, may represent the latter type aging determinants. These proteins are deposited before the birth of a virgin yeast cell. During the subsequent division of this cell, the original protein population remains tightly associated with the mother cortex, while the newly synthesized transporter proteins are deposited mostly into the bud. Thus, the new and old pools of membrane-bound MDR proteins are spatially segregated during yeast asymmetric cell division with the older pool stably inherited by the aging mother. A model based on the observed dynamics of MDR protein inheritance and turnover predicted a decline in MDR activity as the mother cell advances in replicative age. As MDR proteins play crucial roles in cellular metabolism, detoxification and stress response, their collective decline may lead to fitness loss at an advance age. Supporting this hypothesis, mutants lacking certain MDR genes exhibited a reduced replicative lifespan (RLS), while introduction of only one extra copy of these MDR genes extended RLS.
The Bacillus subtilis Pho signal transduction network, which regulates the cellular response to phosphate starvation, integrates the activity of three signal transduction systems to regulate the level of the Pho response. This signal transduction network includes a positive feedback loop between the PhoP/PhoR and ResD/ResE two-component systems. Within this network, ResD is responsible for 80% of the Pho response. To date, the role of ResD in the generation of the Pho response has not been understood. Expression of two terminal oxidases requires ResD function, and expression of at least one terminal oxidase is needed for the wild-type Pho response. Previously, our investigators have shown that strains bearing mutations in resD are impaired for growth and acquire secondary mutations which compensate for the loss of the a-type terminal oxidases by allowing production of cytochrome bd. We report here that the expression of cytochrome bd in a ⌬resDE background is sufficient to compensate for the loss of ResD for full Pho induction. A ctaA mutant strain, deficient in the production of heme A, has the same Pho induction phenotype as a ⌬resDE strain. This demonstrates that the production of a-type terminal oxidases is the basis for the role of ResD in Pho induction. Terminal oxidases affect the redox state of the quinone pool. Reduced quinones inhibit PhoR autophosphorylation in vitro, consistent with a requirement for terminal oxidases for full Pho induction in vivo.The Bacillus subtilis phosphate starvation response (Pho response) is under the control of a complex regulatory network that allows the cell to respond to the level of inorganic phosphate (P i ) in the environment. This system is critical to survival because phosphate is the limiting nutrient in soil (33), the natural environment for B. subtilis.Central to the B. subtilis Pho response is the PhoP/PhoR two-component signal transduction system. The phoPR operon (23, 43) is subject to activation by PhoP under phosphate starvation conditions (34). PhoP/PhoR directly regulates the expression of genes involved in the cellular response to phosphate starvation. The histidine kinase, PhoR, is autophosphorylated in response to an environmental signal and then phosphorylates its cognate response regulator, PhoP. PhoPϳP activates the transcription of the alkaline phosphatases (19), phoA (formerly phoAIV) (20), and phoB (formerly phoAIII) (7); phosphodiesterases, phoD (11), and glpQ (1); a high-affinity phosphate transport system, pstS (37); teichuronic acid synthetic genes (teichuronic acid is a cell wall polymer lacking phosphate), tuaABCDEFGH (27, 46); and a gene encoding a 60-residue peptide of unknown function, ykoL (38). PhoPϳP has been shown to repress the expression of the tagAB and tagDEF genes responsible for the production of teichoic acid (a cell wall polymer containing phosphate) (26). The collective action of these products allows the cell to scavenge extracellular phosphate and to release additional P i from the cell wall.The regulation of the Pho respon...
The phoB gene of Bacillus subtilis encodes an alkaline phosphatase (PhoB, formerly alkaline phosphatase III) that is expressed from separate promoters during phosphate deprivation in a PhoP-PhoR-dependent manner and at stage two of sporulation under phosphate-sufficient conditions independent of PhoP-PhoR. Isogenic strains containing either the complete phoB promoter or individual phoB promoter fusions were used to assess expression from each promoter under both induction conditions. The phoB promoter responsible for expression during sporulation, phoB-P S , was expressed in a wild-type strain during phosphate deprivation, but induction occurred >3 h later than induction of Pho regulon genes and the levels were approximately 50-fold lower than that observed for the PhoPR-dependent promoter, phoB-P V . E E was necessary and sufficient for P S expression in vitro. P S expression in a phoPR mutant strain was delayed 2 to 3 h compared to the expression in a wild-type strain, suggesting that expression or activation of E is delayed in a phoPR mutant under phosphate-deficient conditions, an observation consistent with a role for PhoPR in spore development under these conditions. Phosphorylated PhoP (PhoPϳP) repressed P S in vitro via direct binding to the promoter, the first example of an E E -responsive promoter that is repressed by PhoPϳP. Whereas either PhoP or PhoPϳP in the presence of E A was sufficient to stimulate transcription from the phoB-P V promoter in vitro, roughly 10-and 17-fold-higher concentrations of PhoP than of PhoPϳP were required for P V promoter activation and maximal promoter activity, respectively. The promoter for a second gene in the Pho regulon, ykoL, was also activated by elevated concentrations of unphosphorylated PhoP in vitro. However, because no Pho regulon gene expression was observed in vivo during P i -replete growth and PhoP concentrations increased only threefold in vivo during phoPR autoinduction, a role for unphosphorylated PhoP in Pho regulon activation in vivo is not likely.
The PhoPR two-component system activates or represses Pho regulon genes to overcome a phosphate deficiency. The Pho signal transduction network is comprised of three two-component systems, PhoPR, ResDE, and Spo0A. Activated PhoP is required for expression of ResDE from the resA promoter, while ResD is essential for 80% of Pho induction, establishing a positive feedback loop between these two-component systems to amplify the signal received by the Pho system. The role of ResD in the Pho response is via production of terminal oxidases. Reduced quinones inhibit PhoR autophosphorylation in vitro, and it was proposed that the expression of terminal oxidases leads to oxidation of the quinone pool, thereby relieving the inhibition. We show here that the reducing environment generated by dithiothreitol (DTT) in vivo inhibited Pho induction in a PhoR-dependent manner, which is in agreement with our previous in vitro data. A strain containing a PhoR variant, PhoR C303A , exhibited reduced Pho induction and remained sensitive to inhibition by DTT, suggesting that the mechanisms for Pho reduction via PhoR C303A and DTT are different. PhoR and PhoR C303A were similar with regard to cellular concentration, limited proteolysis patterns, rate of autophosphorylation, stability of PhoRϳP, and inhibition of autophosphorylation by DTT. Phosphotransfer between PhoRϳP or PhoR C303A ϳP and PhoP occurred rapidly; most label from PhoRϳP was transferred to PhoP, but only 10% of the label from PhoR C303A ϳP was associated with PhoP, while 90% was released as inorganic phosphate. No difference in PhoPϳP or PhoR autophosphatase activity was observed between PhoR and PhoR C303A that would explain the release of inorganic phosphate. Our data are consistent with a role for PhoR C303 in PhoR activity via stabilization of the phosphoryl-protein intermediate(s) during phosphotransfer from PhoRϳP to PhoP, which is stabilization that is required for efficient production of PhoPϳP.
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