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Protein export from the endoplasmic reticulum (ER) depends on the interaction between a signal motif on the cargo and a cargo recognition site on the coatomer protein complex II. A hydrophobic sequence in the N terminus of the bovine anion exchanger 1 (AE1) anion exchanger facilitated the ER export of human AE1Δ11, an ER-retained AE1 mutant, through interaction with a specific Sec24 isoform. The cell surface expression and N-glycan processing of various substitution mutants or chimeras of human and bovine AE1 proteins and their Δ11 mutants in HEK293 cells were examined. The N-terminal sequence (V/L/F)X(I/L)X(M/L), 26VSIPM30 in bovine AE1, which is comparable with ΦXΦXΦ, acted as the ER export signal for AE1 and AE1Δ11 (Φis a hydrophobic amino acid, and X is any amino acid). The AE1-Ly49E chimeric protein possessing the ΦXΦXΦ motif exhibited effective cell surface expression and N-glycan maturation via the coatomer protein complex II pathway, whereas a chimera lacking this motif was retained in the ER. A synthetic polypeptide containing the N terminus of bovine AE1 bound the Sec23A-Sec24C complex through a selective interaction with Sec24C. Co-transfection of Sec24C-AAA, in which the residues 895LIL897 (the binding site for another ER export signal motif IXM on Sec24C and Sec24D) were mutated to 895AAA897, specifically increased ER retention of the AE1-Ly49E chimera. These findings demonstrate that the ΦXΦXΦsequence functions as a novel signal motif for the ER export of cargo proteins through an exclusive interaction with Sec24C. [ABSTRACT FROM AUTHOR]

During neurite outgrowth, Rho small G protein activity is spatiotemporally regulated to organize the neurite sprouting, extension, and branching. We have previously identified a potent Rho GTPase-activating protein (GAP), RA-RhoGAP, as a direct downstream target of Rap1 small G protein in the neurite outgrowth. In addition to the Ras-associating (RA) domain for Rap1 binding, RA-RhoGAP has the pleckstrin homology (PH) domain for lipid binding. Here, we showed that phosphatidic acid (PA) bound to the PH domain and enhanced GAP activity for Rho. RA-RhoGAP induced extension of neurite in a diacylglycerol kinase-mediated synthesis of the PA-dependent manner. Knockdown of RA-RhoGAP reduced the diacylglycerol kinase-induced neurite extension. In contrast to the effect of the RA domain, the PH domain was specifically involved in the neurite extension, not in the sprouting and branching. These results indicate that PA and Rap1 cooperatively regulate RA-RhoGAP activity for promoting neurite outgrowth. [ABSTRACT FROM AUTHOR]

The article presents a study which investigates the formation of bovine anion exchanger R664X AE1 mutant in HEK293 cells. It mentions the use of immunofluorescence microscopy in the analysis of HEK293 cells which are transfected in different plasmid constructs by using TransIt-LT1 reagent. Results show that the endoplasmic reticulum (ER) retardation of R664X AE1 is changed and the AE1 mutant is forcedly dislocated in the ER that lead to the forced formation with cystic fibrosis transmembrane.

Neurotransmitter release is triggered by Ca2+ binding to a low affinity Ca2+ sensor, mostly synaptotagmin-1, which catalyzes SNARE-mediated synaptic vesicle fusion. Tomosyn negatively regulates Ca2+-dependent neurotransmitter release by sequestering target SNAREs through the C-terminal VAMP-like domain. In addition to the C terminus, the N-terminal WD40 repeats of tomosyn also have potent inhibitory activity toward Ca2+-dependent neurotransmitter release, although the molecular mechanism underlying this effect remains elusive. Here, we show that through its N-terminal WD40 repeats tomosyn directly binds to synaptotagmin-1 in a Ca2+-dependent manner. The N-terminal WD40 repeats impaired the activities of synaptotagmin-1 to promote SNARE complex-mediated membrane fusion and to bend the lipid bilayers. Decreased acetylcholine release from N-terminal WD40 repeat-microinjected superior cervical ganglion neurons was relieved by microinjection of the cytoplasmic domain of synaptotagmin-1. These results indicate that, upon direct binding, the N-terminal WD40 repeats negatively regulate the synaptotagmin-1-mediated step of Ca2+-dependent neurotransmitter release. Furthermore, we show that synaptotagmin-1 binding enhances the target SNARE-sequestering activity of tomosyn. These results suggest that the interplay between tomosyn and synaptotagmin-1 underlies inhibitory control of Ca2+-dependent neurotransmitter release. [ABSTRACT FROM AUTHOR]