An Stomatin, Prohibitin, Flotillin, and HflK/C-Domain Protein Required to Link the Phage-Shock Protein to the Membrane in Bacillus subtilis

Membrane surveillance and repair is of utmost importance to maintain cellular integrity and allow cellular life. Several systems detect cell envelope stress caused by antimicrobial compounds and abiotic stresses such as solvents, pH-changes and temperature in bacteria. Proteins containing an Stomatin, Prohibitin, Flotillin, and HflK/C (SPFH)-domain, including bacterial flotillins have been shown to be involved in membrane protection and membrane fluidity regulation. Here, we characterize a bacterial SPFH-domain protein, YdjI that is part of a stress induced complex in Bacillus subtilis. We show that YdjI is required to localize the ESCRT-III homolog PspA to the membrane with the help of two membrane integral proteins, YdjG/H. In contrast to classical flotillins, YdjI resides in fluid membrane regions and does not enrich in detergent resistant membrane fractions. However, similarly to FloA and FloT from B. subtilis, deletion of YdjI decreases membrane fluidity. Our data reveal a hardwired connection between phage shock response and SPFH proteins.

Evolutionary conservation of intrinsically unstructured regions in slit-diaphragm proteins

Vertebrate kidneys contribute to homeostasis by regulating electrolyte, acid-base balance, removing toxic metabolites from blood, and preventing protein loss into the urine. Glomerular podocytes constitute the blood-urine barrier, and podocyte slit-diaphragm (SD), a modified tight junction, contributes to the glomerular permselectivity. Nephrin, KIRREL1, podocin, CD2AP, and TRPC6 are crucial members of the SD that interact with each other and contribute to the SD’s structural and functional integrity. This study analyzed the distribution of these five essential SD proteins across the organisms for which the genome sequence is available. We found a diverse distribution of nephrin and KIRREL1 ranging from nematodes to higher vertebrates, whereas podocin, CD2AP, and TRPC6 are restricted to the vertebrates. Among invertebrates, nephrin and its orthologs consist of more immunoglobulin-3 domains, whereas in the vertebrates, CD80-like C2-set domains are predominant. In the case of KIRREL1 and its orthologs, more Ig domains were observed in invertebrates than vertebrates. Src Homology-3 (SH3) domain of CD2AP and SPFH domain of podocin are highly conserved among vertebrates. TRPC6 and its orthologs had conserved ankyrin repeats, TRP, and ion transport domains, except Chondrichthyes and Echinodermata, which do not possess the ankyrin repeats. Intrinsically unstructured regions (IURs) are conserved across the SD orthologs, suggesting IURs importance in the protein complexes that constitute the slit-diaphragm. For the first time, a study reports the evolutionary insights of vertebrate SD proteins and their invertebrate orthologs.

Effect of 17β-estradiol on a Human Vaginal Lactobacillus Crispatus Strain

Lactobacillis and estrogens are essential in vaginal homeostasis. In the present study, we investigated the potential direct effect of 17β-estradiol on a vaginal strain of Lactobacillus cristapus, the major bacterial species of the vaginal microbiota. 17β-estradiol (10− 6 to 10− 10 M) has no effect on the growth of L. crispatus but markedly affects its membrane dynamic. This effect appears adaptative and coherent with a signal transduction process. The surface polarity and aggregation potential of the bacteria remains unchanged after exposure to 17β-estradiol but its mean size is significantly reduced. 17β-estradiol also promotes biosurfactant production by L. crispatus and adhesion to vaginal VK2/E6E7 cells. Conversely, the biofilm formation activity of the bacterium is marginally affected. A potential 17β-estradiol binding site was identified by bioinformatics in L. crispatus as the membrane lipid rafts associated SPFH domain containing protein. Taken together, this study reveals that 17β-estradiol can exert direct effects on L. crispatus. This process might be of importance in the vaginal environment physiology by promoting adhesion of Lactobacilli to the mucosa and providing protection against pathogens.

Molecular Evolution of Podocyte Slit-diaphragm Proteins

Vertebrates kidneys contribute to the homeostasis by regulating electrolyte, acid-base balance, and prevent protein loss into the urine. Glomerular podocytes constitute blood-urine barrier and podocyte slit-diaphragm, a modified tight junction contributes to the glomerular permselectivity. Nephrin, podocin, CD2AP, and TRPC6 are considered to be crucial members, which largely interact with each other and contribute to the structural and functional integrity of the slit-diaphragm. In this study, we analyzed the distribution of these four-key slit-diaphragm proteins across the organisms for which the genome sequence is available. We found that nephrin has a diverse distribution ranging from nematodes to higher vertebrates whereas podocin, CD2AP, and TRPC6 are predominantly restricted to the vertebrates. In the invertebrates nephrin and its orthologs consist of more immunoglobulin-3 and immunoglobulin-5 domains when compared to the vertebrates wherein, CD80-like C2-set Ig2 domains were predominant. Src Homology-3 (SH3) domain of CD2AP and SPFH domain of podocin are highly conserved among vertebrates. Although the majority of the TRPC6 and its orthologs had conserved ankyrin repeats, TRP, and ion transport domains, the orthologs of TRPC6 present in Rhincodon typus and Acanthaster planci do not possess the ankyrin repeats. Intrinsically unstructured regions (IURs), which are considered to contribute to the interactions among these proteins are largely conserved among orthologs of these proteins, suggesting the importance of IURs in the protein complexes that constitute slit-diaphragm. This study for the first time reports the evolutionary insights of vertebrate slit-diaphragm proteins and its invertebrate orthologs.

RAB31 marks and controls an ESCRT-independent exosome pathway

Exosomes are generated within the multivesicular endosomes (MVEs) as intraluminal vesicles (ILVs) and secreted during the fusion of MVEs with the cell membrane. The mechanisms of exosome biogenesis remain poorly explored. Here we identify that RAB31 marks and controls an ESCRT-independent exosome pathway. Active RAB31, phosphorylated by epidermal growth factor receptor (EGFR), engages flotillin proteins in lipid raft microdomains to drive EGFR entry into MVEs to form ILVs, which is independent of the ESCRT (endosomal sorting complex required for transport) machinery. Active RAB31 interacts with the SPFH domain and drives ILV formation via the Flotillin domain of flotillin proteins. Meanwhile, RAB31 recruits GTPase-activating protein TBC1D2B to inactivate RAB7, thereby preventing the fusion of MVEs with lysosomes and enabling the secretion of ILVs as exosomes. These findings establish that RAB31 has dual functions in the biogenesis of exosomes: driving ILVs formation and suppressing MVEs degradation, providing an exquisite framework to better understand exosome biogenesis.

Identification, Localization, and Functional Implications of the Microdomain-Forming Stomatin Family in the Ciliated Protozoan Paramecium tetraurelia

ABSTRACT The SPFH protein superfamily is assumed to occur universally in eukaryotes, but information from protozoa is scarce. In the Paramecium genome, we found only Stomatins, 20 paralogs grouped in 8 families, STO1 to STO8 . According to cDNA analysis, all are expressed, and molecular modeling shows the typical SPFH domain structure for all subgroups. For further analysis we used family-specific sequences for fluorescence and immunogold labeling, gene silencing, and functional tests. With all family members tested, we found a patchy localization at/near the cell surface and on vesicles. The Sto1p and Sto4p families are also associated with the contractile vacuole complex. Sto4p also makes puncta on some food vacuoles and is abundant on vesicles recycling from the release site of spent food vacuoles to the site of nascent food vacuole formation. Silencing of the STO1 family reduces mechanosensitivity (ciliary reversal upon touching an obstacle), thus suggesting relevance for positioning of mechanosensitive channels in the plasmalemma. Silencing of STO4 members increases pulsation frequency of the contractile vacuole complex and reduces phagocytotic activity of Paramecium cells. In summary, Sto1p and Sto4p members seem to be involved in positioning specific superficial and intracellular microdomain-based membrane components whose functions may depend on mechanosensation (extracellular stimuli and internal osmotic pressure).