Enzymes of the ATP-independent Deg serine endopeptidase family are very flexible with regard to their substrate specificity. Some family members cleave only one substrate, while others act as general proteases on unfolded substrates. The proteolytic activity of Deg proteases is regulated by PDZ protein interaction domains. Here we characterized the HhoA protease from Synechocystis sp. strain PCC 6803 in vitro using several recombinant protein constructs. The proteolytic activity of HhoA was found to increase with temperature and basic pH and was stimulated by the addition of Mg 2؉ or Ca 2؉ . We found that the single PDZ domain of HhoA played a critical role in regulating protease activity and in the assembly of a hexameric complex. Deletion of the PDZ domain strongly reduced proteolysis of a sterically challenging resorufin-labeled casein substrate, but unlabeled ␤-casein was still degraded. Reconstitution of the purified HhoA with total membrane proteins isolated from Synechocystis sp. wild-type strain PCC 6803 and a ⌬hhoA mutant resulted in specific degradation of selected proteins at elevated temperatures. We concluded that a single PDZ domain of HhoA plays a critical role in defining the protease activity and oligomerization state, combining the functions that are attributed to two PDZ domains in the homologous DegP protease from Escherichia coli. Based on this first enzymatic study of a Deg protease from cyanobacteria, we propose a general role for HhoA in the quality control of extracytoplasmic proteins, including membrane proteins, in Synechocystis sp. strain PCC 6803.Proteolysis is an essential process in every living cell and is involved in protein quality control (45), as well as in regulation of diverse cellular events (11). In the cyanobacterium Synechocystis sp. strain PCC 6803, which is a widely used model system for studying photosynthesis and acclimation to abiotic stresses, 77 proteases are encoded in the genome (http://merops.sanger .ac.uk/). Three genes encode ATP-independent serine endopeptidases belonging to the Deg (HtrA) family (14,19,21,40). The protease domain of these enzymes harbors the hallmark catalytic triad consisting of His, Asp, and Ser responsible for the proteolytic activity and may additionally confer a chaperone function (16,42). Deg proteases usually contain one or two protein-protein interaction domains of the PDZ type C terminal of the protease domain (7). Studies of Escherichia coli DegP (also designated HtrA), E. coli DegS (also designated HhoB), and human HtrA2 showed that PDZ domains regulate the proteolytic activity (16,18,27,38,42,46), are involved in the formation of homooligomeric complexes (16,18,38), and play a role in substrate recognition (7,16,23,35).In prokaryotes, Deg proteases are usually located in the periplasm and are implicated in the response to a variety of stresses, such as heat (3, 30, 33), oxidative stress (3, 47), and high-light stress (3, 39). Deg proteases are also essential for virulence in several pathogenic bacteria (33,47). An intriguing fea...

Human granulocytes express several glycoproteins of the CEACAM family. One family member, CEACAM3, operates as a single-chain phagocytic receptor, initiating the detection, internalization, and destruction of a limited set of gram-negative bacteria. In contrast, the function of CEACAM4, a closely related protein, is completely unknown. This is mainly a result of a lack of a specific ligand for CEACAM4. By generating chimeric proteins containing the extracellular bacteria-binding domain of CEACAM3 and the transmembrane and cytoplasmic part of CEACAM4 (CEACAM3/4) we demonstrate that this chimeric receptor can trigger efficient phagocytosis of attached particles. Uptake of CEACAM3/4-bound bacteria requires the intact ITAM of CEACAM4, and this motif is phosphorylated by Src family PTKs upon receptor clustering. Furthermore, SH2 domains derived from Src PTKs, PI3K, and the adapter molecule Nck are recruited and associate directly with the phosphorylated CEACAM4 ITAM. Deletion of this sequence motif or inhibition of Src PTKs blocks CEACAM4-mediated uptake. Together, our results suggest that this orphan receptor of the CEACAM family has phagocytic function and prompt efforts to identify CEACAM4 ligands.