Localization of general and regulatory proteolysis in Bacillus subtilis cells

Abstract

SummaryProtein degradation mediated by ATP-dependent proteases, such as Hsp100/Clp and related AAA+ proteins, plays an important role in cellular protein homeostasis, protein quality control and the regulation of, e.g. heat shock adaptation and other cellular differentiation processes. ClpCP with its adaptor proteins and other related proteases, such as ClpXP or ClpEP of Bacillus subtilis, are involved in general and regulatory proteolysis. To determine if proteolysis occurs at specific locations in B. subtilis cells, we analysed the subcellular distribution of the Clp system together with adaptor and general and regulatory substrate proteins, under different environmental conditions. We can demonstrate that the ATPase and the proteolytic subunit of the Clp proteases, as well as the adaptor or substrate proteins, form visible foci, representing active protease clusters localized to the polar and to the mid-cell region. These clusters could represent a compartmentalized place for protein degradation positioned at the pole close to where most of the cellular protein biosynthesis and also protein quality control are taking place, thereby spatially separating protein synthesis and degradation.

Genetic and cytological evidence that heterocyst patterning is regulated by inhibitor gradients that promote activator decay

“…Because both PatS and HetN contain the RGSGR peptide motif and Golden's group has shown that confinement of PatS to source cells prevents proper heterocyst patterning (7), it seems likely that portions of PatS and HetN that include the RGSGR motif constitute diffusible inhibitory signals. The foci of HetR-GFP fluorescence elicited by RGSGR peptide are strikingly similar to those recently observed when various Clp proteases and their substrates were fused to GFP in Bacillus subtilis and Caulobacter crescentus (17)(18)(19), suggesting that PatS-and HetN-dependent signals may target HetR for transport to, and degradation by, cellular proteases located at discreet positions in the cell. PatSand HetN-dependent decay of HetR is distinct from previously reported HetR-autoproteolysis or HetF-dependent degradation as evidenced by decay of HetR S179N , which is not autoproteolytic (15), and decay of HetR in a hetF-mutant background.…”

Global impact of protein arginine phosphorylation on the physiology of Bacillus subtilis

“…We attempted to identify phosphorylation on protein arginine residues in vivo in a B. subtilis wild-type strain with various proteomic approaches, but failed to detect it. However, the YwlE phosphatase had been identified as the cognate McsB phosphatase in vitro and was shown to antagonize the activity of McsB in vivo (8,9,(11)(12)(13). This observation strongly suggests that YwlE may act as a general protein arginine phosphatase so that protein phosphorylation on arginine residues could be more stable and enriched in an ywlE deletion strain.…”

“…As noted above, it was reported that YwlE is the cognate phosphatase for the McsB kinase in vitro and also antagonizes McsB activity in vivo (8,9,(11)(12)(13). Consequently, we wondered whether McsB is solely responsible for the arginine phosphorylations detected in the ywlE mutant (Table 1 and Fig.…”

Activity Control of the ClpC Adaptor McsB in Bacillus subtilis

“…Recently, it was reported that the YwlE phosphatase antagonizes McsB in vivo (9,16,22). Accordingly, we were interested in whether YwlE contributes to the regulation of McsB activity.…”