SummaryThe ATP-dependent protease Clp plays important roles in the cell's protein quality control system and in the regulation of cellular processes. In Corynebacterium glutamicum , the levels of the proteolytic subunits ClpP1 and ClpP2 as well as of the corresponding mRNAs were drastically increased upon deletion of the clpC gene, coding for a Clp ATPase subunit. We identified a regulatory protein, designated ClgR, binding to a common palindromic sequence motif in front of clpP1P2 as well as of clpC . Deletion of clgR in the D D D D clpC background completely abolished the increased transcription of both operons, indicating that ClgR activates transcription of these genes. ClgR activity itself is probably controlled via ClpC-dependent regulation of its stability, as ClgR is only present in D D D D clpC and not in wild-type cells, whereas the levels of clgR mRNA are comparable in both strains. clpC , clpP1P2 and clgR expression is induced upon severe heat stress, however, independently of ClgR. Identification of the heat-responsive transcriptional start sites in front of these genes revealed the presence of sequence motifs typical for s s s s ECF -dependent promoters. The ECF sigma factor s s s s H could be identified as being required for transcriptional activation of clpC , clpP1P2 and clgR in response to severe heat stress. A second heat-responsive but s s s s H -independent promoter in front of clgR could be identified that is subject to negative regulation by the transcriptional repressor HspR. Taken together, these results show that clpC and clpP1P2 expression in C. glutamicum is subject to complex regulation via both independent and hierarchically organized pathways, allowing for the integration of multiple environmental stimuli. Both the ClgR-and s s s s H -dependent regulation of clpC and clpP1P2 expression appears to be conserved in other actinomycetes. IntroductionProteolysis in bacterial cells is mainly performed by ATPdependent proteases. Together with several chaperones, i.e. DnaKJ-GrpE and ClpB, these proteases are integral parts of the cell's protein quality control system, which is responsible for clearing the cell of non-functional proteins (for recent reviews, see Wickner et al ., 1999;Dougan et al ., 2002). Most of these proteases also perform important regulatory functions by controlling the availability of transcriptional regulators, enzymes and other proteins via conditional degradation (for a recent review, see Jenal and Hengge-Aronis, 2003). Of these proteases, Clp has been most extensively studied both mechanistically and functionally. The Clp holoenzyme consists of two separate and functionally distinct subunits. The proteolytic subunits, ClpP, perform the actual hydrolysis of substrates. However, their active sites are buried within the cavity of the so-called proteolytic core formed by 14 ClpP subunits. Therefore, hexamers of ATPase subunits (ClpA, ClpC or ClpX), which are members of the Clp/Hsp100 superfamily (Schirmer et al ., 1996) and associate with the core, are required in orde...
Annexin 2 is a Ca2+-regulated membrane protein and an F-actin-binding protein enriched at actin assembly sites both, on the plasma membrane and on endosomal vesicles. Here, we identify annexin 2 as a phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2)-interacting protein, thereby explaining this specific membrane association. Using the pleckstrin-homology (PH) domain of phospholipase Cδ1 fused to yellow fluorescent protein as a marker for PtdIns(4,5)P2, we show that annexin 2 and its ligand p11 (S100A10) are targeted to sites of PtdIns(4,5)P2 enrichment where F-actin accumulates. At the plasma membrane, adhesion of pedestal-forming enteropathogenic Escherichia coli induces a recruitment of 1-phosphatidylinositol-4-phosphate 5-kinase (PtdIns4P 5-kinase) and an enrichment of PtdIns(4,5)P2 and annexin 2-p11 at sites of bacterial adhesion. Induction of PtdIns(4,5)P2-enriched ruffles and PtdIns(4,5)P2-positive, actin-coated vacuoles by Arf6-mediated activation of PtdIns4P 5-kinase also leads to a concomitant accumulation of the annexin 2-p11 complex and the PH domain. Binding studies with immobilized phosphoinositides and phosphoinositide-containing liposomes reveal that the purified annexin 2-p11 complex directly and specifically binds to PtdIns(4,5)P2 with an affinity comparable to that of the PH domain of phospholipase Cδ1. Experiments using individual subunits identify annexin 2 as the PtdIns(4,5)P2-binding entity. Thus, the direct interaction of annexin 2 with PtdIns(4,5)P2 is a means of specifically recruiting the annexin 2-p11 complex to sites of membrane-associated actin assembly.
Cell adhesion and motility depend on dynamic responses of the cytoskeleton that are regulated by the precise coordination and integration of multiple signaling cascades transmitting various extracellular signals. Central to many of these processes is a remodelling of the actin cytoskeleton, which requires the coordinated activity of specific actin-binding proteins together with numerous additional cytoskeletal and signaling molecules, such as kinases and GTPases. Extracellular signals that trigger changes in the actin cytoskeleton resulting in altered cell adhesion and motility have been described, and the signaling receptors involved are known. In many cases these belong to the family of receptor tyrosine kinases (RTKs) with prominent examples being the receptors for epidermal growth factor, ephrins and insulin (for reviews, see Heldin, 1996;Schlessinger, 2000;Ullrich and Schlessinger, 1990). Owing to its importance in cellular adhesion and cell motility, RTK signaling and its deregulation are also critically involved in tumorigenesis.Activation of the insulin receptor, in addition to initiating a wide range of metabolic and nuclear responses, elicits changes in the Factin distribution, manifesting in increased membrane ruffling, dispersion of focal adhesion proteins and a loss of cell-substratum contact (Moller et al., 1995;Tsakiridis et al., 1999). A number of substrates of the insulin receptor participating in the regulation of these responses have been described, including the insulin receptor substrate p53, a multidomain scaffolding protein implicated in filopodium and lamellipodium formation (Govind et al., 2001;Miki et al., 2000). However, many signaling intermediates and the respective pathways involved in mediating cell morphology changes and loss in substrate adhesion following insulin receptor stimulation remain to be elucidated.Cells overexpressing the insulin receptor have been introduced as model systems to study tyrosine-kinase-based signaling because
The Ca 2؉ -and lipid-binding protein annexin 2, which resides in a tight heterotetrameric complex with the S100 protein S100A10 (p11), has been implicated in the structural organization and dynamics of endosomal membranes. To elucidate the function of annexin 2 and S100A10 in endosome organization and trafficking, we used RNA-mediated interference to specifically suppress annexin 2 and S100A10 expression. Down-regulation of both proteins perturbed the distribution of transferrin receptor-and rab11-positive recycling endosomes but did not affect uptake into sorting endosomes. The phenotype was highly specific and could be rescued by reexpression of the N-terminal annexin 2 domain or S100A10 in annexin 2-or S100A10-depleted cells, respectively. Whole-mount immunoelectron microscopy of the aberrantly localized recycling endosomes in annexin 2/S100A10 down-regulated cells revealed extensively bent tubules and an increased number of endosome-associated clathrin-positive buds. Despite these morphological alterations, the kinetics of transferrin uptake and recycling was not affected to a significant extent, indicating that the proper positioning of recycling endosomes is not a rate-limiting step in transferrin recycling. The phenotype generated by this transient loss-of-protein approach shows for the first time that the annexin 2/S100A10 complex functions in the intracellular positioning of recycling endosomes and that both subunits are required for this activity.
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