Translocation domains

Fig. 7. Two possible interpretations of the transitions of the translocator domain detected by the kinetics of binding to ISO membranes at 4°C. (A) Translocation. The states not in contact with the external medium are the states in contact with the internal medium. (B) Occlusion. The binding sites can be in a state where they are not accessible from either side of the membrane. The spots represent the substrate molecule, cyt and per represent the cytoplasmic and periplasmic side of the enzyme, respectively.

Clark, J. D., Lin, L.-L., Kriz, R. W. etal. A novel arachidonic acid-selective cytosolic PLA2 contains a Ca2+-dependent translocation domain with homology to PKC and GAP Cell 65 1043-1051,1991. 

Figure 1 The mode of action for bacterial AB-type exotoxins. AB-toxins are enzymes that modify specific substrate molecules in the cytosol of eukaryotic cells. Besides the enzyme domain (A-domain), AB-toxins have a binding/translocation domain (B-domain) that specifically interacts with a cell-surface receptor and facilitates internalization of the toxin into cellular transport vesicles, such as endosomes. In many cases, the B-domain mediates translocation of the A-domain into the cytosol by pore formation in cellular membranes. By following receptor-mediated endocytosis, AB-type toxins exploit normal vesicle traffic pathways into cells. One type of toxin escapes from early acidified endosomes (EE) into the cytosol, thus they are referred to as short-trip-toxins . In contrast, the long-trip-toxins take a retrograde route from early endosomes (EE) through late endosomes (LE), trans-Golgi network (TGN), and Golgi apparatus into the endoplasmic reticulum (ER) from where the A-domains translocate into the cytosol to modify specific substrates.

BoNTs (150 kDa) consist of two polypeptide chains the heavy chain (HC, 100 kDa) and the light chain (LC, 50 kDa), linked with disulfide and non-covalent bonds. The amine end of the LC is responsible for intraneural enzymatic activity. The HC contains a membrane translocation domain (a 50 kDa amino-terminal polypeptide) and a receptor-binding part (a 50 kDa carboxy-terminal polypeptide) (DasGupta, 1990 Krieglstein et ah, 1994). BoNT/A forms dimers, trimers, and bigger structures. BoNT/E generally has a monomer structure, but sometimes forms dimers. BoNT/B is a dimer (Ledoux et ah, 1994). 

Membrane translocation domains have been identified in toxins and viruses and derived from signal sequences of secreted proteins. When derived from a signal seqnence the translocation domain contains hydrophobic sequences [146-148] while the toxin and viral translocation domains contain mostly basic residues [149,150]. 

In terms of targeting, membrane translocation domains lack specificity for particnlar cells or tissues. Therefore, these domains shonld be combined with targeting domains snch as those discussed in the previous section. In snch a constrnct, the targeting domain wiU ensnre a rapid accumulation at the surface of a specific cell type and the translocation domain will facilitate entry into the cytosol of the target cells. 

The N-terminal portion of the 522-residue polypeptide chain of colicin El appears to be required for transport into the membrane and the central part for binding to the receptor the channelforming property is characteristic of the C-terminal region.k A similar organization has been established for the smaller colicin N translocation domain, (residues 1-66), receptor domain, (residues 67-182), and pore-forming domain (residues 183-387). 

When the pumps are in the active Ei conformation, Ca2+ appears to have access to these high affinity sites phosphorylation at the (remote) catalytic site will then lead to long-range-transmitted conformational alterations in the geometry of the Ca2+ translocation domain, resulting in Ca2+ ion occlusion and finally, after the E2 conformation has been attained, in exposure of the ion(s) to the trans side. 

A1 and concanamycin A—two established V-ATPase inhibitors.163 This observation was confirmed by both in vitro enzyme assays and in vivo activity studies in mutant yeast. These studies indicated that, while the benzolactone enamides were potent V-ATPase inhibitors, a profound selectivity occurred between the mammalian and fungal enzymes. Subsequent studies by De Brabander determined that salicylihalamide A binds irreversibly to the trans-membranous proton-translocating domain via A-acyliminium chemistry.164... 

Clark JD, Lin L, Kriz R (Other Authors ). (1991). A novel arachidmiic acid-selective cytosolic PLA, contains a Ca -dependent translocation domain with homology to PKC and GAP. Cell 65,1043-1051. 

Unlike diphtheria toxin, little is known about the structures required for the translocation of the enzymatic subunit of PT. In diphtheria toxin and Pseudomonas aeruginosa exotoxin A, the B moiety can be clearly subdivided into two distinct domains, one responsible for receptor binding, composed essentially of (3 sheets, and one responsible for translocation of the A subunits, essentially composed of a helices (Allured etal., 1986 Choe etal., 1992). There is no clear translocation domain in PT, and much less is known about the internalization step of PT, compared to diphtheria toxin and exotoxin A. 

Fusion proteins have been constructed from peptide epitopes from influenza A antigens and the binding and translocation domains of Pseudomonas exotoxin A (Donelly ef al., 1993). When target cells were incubated with these fusion proteins, and subsequently exposed to cytotoxic T lymphocytes (CTLs) specific for the relevant epitopes, a CTL mediate lysis of the target cells was observed. These experiments suggest that the translocation machinery supplied by protein toxins may be useful tools for bringing peptides into cells for presentation via the major histocompatibility class I (MHC I) system. It should be noted that no direct evidence was provided that the translocation occurred by the toxin pathway, and it cannot be excluded that the toxin was only instrumental in accumulating the peptide on the surface of the cells and in the endocytic pathway. 

The HSV-1 virion is approximately 20 run in diameter and consists of four components envelope, tegument, capsid, and viral genome. The envelope is derived from the cellular membrane and contains approximately 12 viral glycoproteins essential for viral entry. The tegument is the protein layer between the capsid and the envelope and contains at least 10 viral proteins, including VP 16 (essential for transactivation and virion envelopment), VP22 (membrane translocation domain), and virion host shut off (vhs) protein. The capsid consists of 7 viral proteins and contains the linear dsDNA genome, which is 152 kb in size. 

PE is a single-chain protein with a molecular mass of 66 kDa composed of three distinct domains (Fig. 2). In the PE protein, domain I (1-252) binds to the PE receptor on normal animal cells, which has been identified as the a2-macroglobulin receptor. Domain II (253-364) mediates translocation of domain III (400-613) into the cytosol. The translocation domain contains a proteolytic cleavage site within a disulfide loop, which, after proteolytic cleavage, leaves the cell-binding site (I) and translocation domain (II) bound to the catalytic/toxic site (III) by a disulfide bond (Fig. 2). Following reduction of this bond in the cytosol, the ADP-ribosylation activity of domain III inactivates elongation factor (EF2) and causes inhibition of protein synthesis and cell death. 

Leamon, C. P., Pastan, I., and Low, P. S. (1993) Cytotoxicity of folate-pseudonomas exotoxin conjugates towards tumor cells. Contribution of translocation domain. J. Biol. Chem. 25, 24,847-24,854. 

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