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Members of the ATP-binding cassette (ABC) transporter superfamily translocate a broad spectrum of chemically diverse substrates. While their eponymous ATP-binding cassette in the nucleotide-binding domains (NBDs) is highly conserved, their transmembrane domains (TMDs) forming the translocation pathway exhibit distinct folds and topologies, suggesting that during evolution the ancient motor domains were combined with different transmembrane mechanical systems to orchestrate a variety of cellular processes. In recent years, it has become increasingly evident that the distinct TMD folds are best suited to categorize the multitude of ABC transporters. We therefore propose a new ABC transporter classification that is based on structural homology in the TMDs., (© 2020 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

CysA, the ATPase subunit of a putative sulfate ATP-binding cassette transport system of the gram-positive thermoacidophilic bacterium Alicyclobacillus acidocaldarius, was structurally characterized at a resolution of 2.0 Angstroms in the absence of nucleotides. In line with previous findings on ABC-ATPases the structures of the two monomers (called CysA-1 and CysA-2) in the asymmetric unit differ substantially in the arrangement of their individual (sub)domains. CysA-2 was found as a physiological dimer composed of two crystallographically related monomers that are arranged in an open state. Interestingly, while the regulatory domain of CysA-2 packs against its opposing domain that of CysA-1 undergoes a conformational change and, in the dimer, would interfere with the opposing monomer thereby preventing solute translocation. Whether this conformational state is used for regulatory purposes will be discussed.

Rapidly generated high-titer Semliki Forest virus (SFV) vectors can infect numerous mammalian cell lines and primary cell cultures, and result in high levels of transgene expression. SFV-based expression of transmembrane receptors has been characterized by specific ligand-binding activity and functional responses. Adaptation of the SFV technology for mammalian suspension cultures has allowed the production of hundreds of milligrams of recombinant receptor for purification and structural studies. The same SFV stock solutions used for the infection of mammalian cells in culture have also been successfully applied for efficient transgene expression in organotypic hippocampal slices, as well as in vivo in rodent brain.

The molecular mechanism(s) by which Bcl-2 regulates apoptosis is poorly understood. Bcl-2 suppresses apoptosis by inhibiting calcium activation of the permeability transition of mitochondria. In this patch-clamp study, overexpression of Bcl-2 in mitochondria of cultured cells suppressed calcium activation of a high conductance channel that may underlie the permeability transition. All other single channel parameters were identical when multiple conductance channel activities of mitochondria from control and Bcl-2 overexpressing cells were compared. Bcl-2 forms channels in artificial membranes; however, no novel channel activities could be linked to Bcl-2 overexpression, suggesting Bcl-2 does not form channels in native inner membranes of mitochondria.

The growth suppressor protein p53 plays a main part in cellular growth control. Two of its key functions are sequence specific DNA binding and transactivation. Functions of p53 in growth control are regulated at least in part by its interaction with protein kinases. p53 binds to protein kinase CK2, formerly known as casein kinase 2, and it is phosphorylated by this enzyme. CK2 is composed of two regulating beta-subunits and two catalytic alpha- or alpha'-subunits and the interaction with p53 is mediated by the regulatory beta-subunit of CK2. Recently we showed that the beta-subunit could inhibit the sequence specific DNA binding activity of p53 in vitro. Based on this finding, we asked if a coexpression of the beta-subunit of CK2 with p53 in mammalian cells could inhibit the DNA binding activity of p53 in a physiological context. We found that the coexpression of the beta-subunit showed the same inhibitory effect as in the previous assays with purified proteins. Then, we investigated the effects of the coexpression of the beta-subunit of CK2 on the transactivation and transrepression activity of p53. We found that transactivation of the mdm2, p21(WAF1/CIP1) and cyclin G promoter was inhibited in three different cell lines whereas transactivation of the bax promoter was not affected in COS1 cells but down-regulated in MCO1 and SaosS138V21 cells. p53 mediated transrepression of the fos promoter was not influenced by coexpression of the CK2 beta-subunit. Taken together we propose a cell type dependent fine regulation of the p53 transactivation function by the CK2 beta-subunit in vivo, which does not affect p53 mediated transrepression.

A gene encoded GADD45 was isolated from rat and revealed four exons along with a p53 binding consensus sequence and a putative AP-1 site in the third intron. This suggests that the rat GADD45 gene is also involved in the p53 signal pathway related to the G1 cell cycle checkpoint. The rat GADD45 mRNA was induced within 30 min in liver and increased as a function of gamma-irradiation. We found that mRNA expression differed substantially in a variety of tissues (brain, liver, kidney, and spleen). The finding of in vivo induction of GADD45 gene may provide insight into the role of GADD45 gene in DNA repair.

Site-directed mutagenesis was used to change four amino acid residues (Q82, P152, L179, H192) in the MalK subunit of S. typhimurium maltose transport system which are highly conserved among members of the ATP-binding cassette (ABC) family. Replacement of H192 caused complete failure to complement the transport defect of a malK strain whereas changes of the other residues resulted in reduced or wild-type activity. The purified mutant proteins exhibited ATPase activity comparable to wild-type MalK.