The interaction of ATP11C-b with ezrin contributes to its polarized localization

ATP11C, a member of the P4-ATPase family, translocates phosphatidylserine and phosphatidylethanolamine at the plasma membrane. We previously revealed that its C-terminal splice variant ATP11C-b exhibits polarized localization in motile cell lines, such as MDA-MB-231 and BaF3. In the present study, we found that the C-terminal cytoplasmic region of ATP11C-b interacts specifically with ezrin. Notably, the LLxY motif in the ATP11C-b C-terminal region is crucial for its interaction with ezrin as well as its polarized localization on the plasma membrane. A constitutively active, C-terminal phosphomimetic mutant of ezrin was colocalized with ATP11C-b in polarized motile cells. ATP11C-b was partially mislocalized in cells depleted of ezrin alone, and exhibited greater mislocalization in cells simultaneously depleted of family members, ezrin, radixin, and moesin (ERM), suggesting that ERM proteins, particularly ezrin, contribute to the polarized localization of ATP11C-b. Further, Atp11c knockout resulted in C-terminally phosphorylated ERM proteins mislocalization, which was restored by exogenous expression of ATP11C-b but not ATP11C-a. These observations together indicate that the polarized localizations of ATP11C-b and the active form of ezrin to the plasma membrane are interdependently stabilized.

Mechanism of β-arrestin recruitment by the μ-opioid G protein-coupled receptor

Agonists to the μ-opioid G protein-coupled receptor (μOR) can alleviate pain through activation of G protein signaling, but they can also induce β-arrestin activation, leading to such side effects as respiratory depression. Biased ligands to μOR that induce G protein signaling without inducing β-arrestin signaling can alleviate pain while reducing side effects. However, the mechanism for stimulating β-arrestin signaling is not known, making it difficult to design optimum biased ligands. We use extensive molecular dynamics simulations to determine three-dimensional (3D) structures of activated β-arrestin2 stabilized by phosphorylated μOR bound to the morphine and D-Ala2,N-MePhe4, Gly-ol]-enkephalin (DAMGO) nonbiased agonists and to the TRV130 biased agonist. For nonbiased agonists, we find that the β-arrestin2 couples to the phosphorylated μOR by forming strong polar interactions with intracellular loop 2 (ICL2) and either the ICL3 or cytoplasmic region of transmembrane (TM6). Strikingly, Gi protein makes identical strong bonds with these same ICLs. Thus, the Gi protein and β-arrestin2 compete for the same binding site even though their recruitment leads to much different outcomes. On the other hand, we find that TRV130 has a greater tendency to bind the extracellular portion of TM2 and TM3, which repositions TM6 in the cytoplasmic region of μOR, hindering β-arrestin2 from making polar anchors to the ICL3 or to the cytosolic end of TM6. This dramatically reduces the affinity between μOR and β-arrestin2.

The extracellular domain of angulin-1 and palmitoylation of its cytoplasmic region are required for angulin-1 assembly at tricellular contacts

Tricellular tight junctions (tTJs) create paracellular barriers at tricellular contacts (TCs), where the vertices of three polygonal epithelial cells meet. tTJs are marked by the enrichment of two types of membrane proteins, tricellulin and angulin family proteins. However, how TC geometry is recognized for tTJ formation remains unknown. In the present study, we examined the molecular mechanism for the assembly of angulin-1 at the TCs. We found that clusters of cysteine residues in the juxtamembrane region within the cytoplasmic domain of angulin-1 are highly palmitoylated. Mutagenesis analyses of the cysteine residues in this region revealed that palmitoylation is essential for localization of angulin-1 at TCs. Consistently, suppression of Asp-His-His-Cys motif–containing palmitoyltransferases expressed in EpH4 cells significantly impaired the TC localization of angulin-1. Cholesterol depletion from the plasma membrane of cultured epithelial cells hampered the localization of angulin-1 at TCs, suggesting the existence of a lipid membrane microdomain at TCs that attracts highly palmitoylated angulin-1. Furthermore, the extracellular domain of angulin-1 was also required for its TC localization, irrespective of the intracellular palmitoylation. Taken together, our findings suggest that both angulin-1's extracellular domain and palmitoylation of its cytoplasmic region are required for its assembly at TCs.

Characterization of PomA periplasmic loop and sodium ion entering in stator complex of sodium-driven flagellar motor

The bacterial flagellar motor is a rotary nanomachine driven by ion flow. The flagellar stator complex, which is composed of two proteins, PomA and PomB, performs energy transduction in marine Vibrio. PomA is a four transmembrane (TM) protein and the cytoplasmic region between TM2 and TM3 (loop2–3) interacts with the rotor protein FliG to generate torque. The periplasmic regions between TM1 and TM2 (loop1–2) and TM3 and TM4 (loop3–4) are candidates to be at the entrance to the transmembrane ion channel of the stator. In this study, we purified the stator complex with cysteine replacements in the periplasmic loops and assessed the reactivity of the protein with biotin maleimide (BM). BM easily modified Cys residues in loop3–4 but hardly labelled Cys residues in loop1–2. We could not purify the plug deletion stator (ΔL stator) composed of PomBΔ41–120 and WT-PomA but could do the ΔL stator with PomA-D31C of loop1–2 or with PomB-D24N of TM. When the ion channel is closed, PomA and PomB interact strongly. When the ion channel opens, PomA interacts less tightly with PomB. The plug and loop1–2 region regulate this activation of the stator, which depends on the binding of sodium ion to the D24 residue of PomB.

Structure of the periplasmic domain of SflA involved in spatial regulation of the flagellar biogenesis of Vibrio reveals a TPR/SLR-like fold

Bacteria have evolved various types of flagellum, an organella for bacterial motility, to adapt to their habitat environments. The number and the spatial arrangement of the flagellum are precisely controlled to optimize performance of each type of the flagellar system. Vibrio alginolyticus has a single sheathed flagellum at the cell pole for swimming. SflA is a regulator protein to prevent peritrichous formation of the sheathed flagellum, and consists of an N-terminal periplasmic region, a transmembrane helix, and a C-terminal cytoplasmic region. Whereas the cytoplasmic region has been characterized to be essential for inhibition of the peritrichous growth, the role of the N-terminal region is still unclear. We here determined the structure of the N-terminal periplasmic region of SflA (SflAN) at 1.9-Å resolution. The core of SflAN forms a domain-swapped dimer with tetratricopeptide repeat (TPR)/Sel1-like repeat (SLR) motif, which is often found in the domains responsible for protein–protein interaction in various proteins. The structural similarity and the following mutational analysis based on the structure suggest that SflA binds to unknown partner protein by SflAN and the binding signal is important for the precise control of the SflA function.