The relationship between endosomal pH and function is well documented in viral entry, endosomal maturation, receptor recycling, and vesicle targeting within the endocytic pathway. However, specific molecular mechanisms that either sense or regulate luminal pH to mediate these processes have not been identified. Herein we describe the use of novel, compartment-specific pH indicators to demonstrate that yeast Nhx1, an endosomal member of the ubiquitous NHE family of Na ؉ /H ؉ exchangers, regulates luminal and cytoplasmic pH to control vesicle trafficking out of the endosome. Loss of Nhx1 confers growth sensitivity to low pH stress, and concomitant acidification and trafficking defects, which can be alleviated by weak bases. Conversely, weak acids cause wild-type yeast to present nhx1⌬ trafficking phenotypes. Finally, we report that Nhx1 transports K ؉ in addition to Na ؉ , suggesting that a single mechanism may responsible for both pH and K ؉ -dependent endosomal processes. This presents the newly defined family of eukaryotic endosomal NHE as novel targets for pharmacological inhibition to alleviate pathological states associated with organellar alkalinization. INTRODUCTIONIt is well established that luminal acidification of the endocytic pathway, including the endosome and lysosome/vacuole, is required for associated cellular function (Mellman et al., 1986;Mellman, 1992). Some examples include ligandreceptor dissociation and recycling of surface receptors, lysosome-mediated protein degradation, H ϩ -driven neurotransmitter loading and pH-dependent recycling of synaptic vesicles (Buckley et al., 2000;Nishi and Forgac, 2002). Similarly, viral pathogen entry and propagation is dependent on the pH gradient across the lumen of the endosome (Harley et al., 2001), and the abnormal lysosomal/endosomal morphologies and associated defective trafficking observed in a subset of lysosomal storage disorders are associated with abnormal changes in luminal pH (Futerman and van Meer, 2004). Pioneering experiments performed by Heuser clearly demonstrated that changes in cellular pH alone severely alter organellar morphology and movement (Heuser, 1989). This phenomenon can be explained by net changes in vesicle trafficking between compartments, as luminal pH can direct vesicle trafficking; thus, elevated pH in the endosome promotes endosome to Golgi vesicle movement (van Weert et al., 1995(van Weert et al., , 1997 also see Nieland et al., 2004). At the molecular level, local increases in pH are believed to be responsible for assembly of vesicle trafficking/sorting machinery in areas of the endosome destined for return to the plasma membrane (Maranda et al., 2001; also see Zeuzem et al., 1992;Aniento et al., 1996). Despite extensive evidence that changes in pH direct trafficking in this pathway, specific molecular mechanisms that control pH itself have not been defined. The ubiquitous Na ϩ /H ϩ exchangers of the NHE family are associated with cellular pH regulation (Orlowski and Grinstein, 2004). Recent phylogenetic analysis of the ...

Endosomal Na؉ /H ؉ exchangers are important for salt and osmotolerance, vacuolar pH regulation, and endosomal trafficking. We show that the C terminus of yeast Nhx1 interacts with Gyp6, a GTPase-activating protein for the Ypt/Rab family of GTPases, and that Gyp6 colocalizes with Nhx1 in the endosomal/prevacuolar compartment (PVC). The gyp6 null mutant exhibits novel phenotypes consistent with loss of negative regulation of Nhx1, including increased tolerance to hygromycin, increased vacuolar pH, and decreased plasma membrane potential. In contrast, overexpression of Gyp6 increases sensitivity to hygromycin, decreases vacuolar pH, and results in a slight missorting of vacuolar carboxypeptidase Y to the cell surface. We conclude that Gyp6 is a negative regulator of Nhx1-dependent trafficking out of the PVC. Taken together with its GTPaseactivating protein-dependent role as a negative regulator of Ypt6-mediated retrograde traffic to the Golgi, we propose that Gyp6 coordinates upstream and downstream events in the PVC to Golgi pathway. Our findings provide a possible molecular link between intraendosomal pH and regulation of vesicular trafficking.

Receptor tyrosine kinases (RTKs) play a pivotal role in the development and function of the cardiovascular system. Ligand-activated RTKs promote numerous downstream signal transduction pathways that lead to vascular permeability, as well as proliferation, migration, and differentiation of vascular endothelia and smooth muscle cells. Ligand binding also promotes internalization of the activated receptors either to downregulate the signaling via degradation of the ligand/receptor complex or to signal from endosomes. However, the outcomes of receptor internalization via clathrin-dependent or caveolar pathways and trafficking mechanisms are incompletely clarified in vascular systems. Activity modulation through endocytosis and vesicular trafficking significantly impacts downstream targets of RTKs such as endothelial nitric oxide synthase (eNOS) and VE-cadherin. RTKs and their associated targets are also transported to the nucleus, where they may directly impact nuclear signaling. Although the nuclear transport pathways are just beginning to be unraveled, it appears that endocytosis and vesicular trafficking are involved. In this review, we discuss the mechanisms by which activated RTKs and the downstream targets eNOS and VE-cadherin may be internalized and transported to various intracellular compartments. How localization and interacting proteins impact protein function and influence signaling is an important theme, as is the potential for modulating signaling through therapeutic targeting of activated receptors and components of the endocytic machinery.

Cytosolic delivery of functional siRNA remains the major challenge to develop siRNA-based therapeutics. Designing clinically safe and effective siRNA transporter to deliver functional siRNA across the plasma and endosomal membrane remains a key hurdle. With the aim of improving endosomal release, we have designed cyclic and linear peptide-based transporters having an Arg-DHis-Arg template. Computational studies show that the Arg-DHis-Arg template is also stabilized by the Arg–His side-chain hydrogen bonding interaction at physiological pH, which dissociates at lower pH. The overall atomistic interactions were examined by molecular dynamics simulations, which indicate that the extent of peptide_siRNA assembly formation depends greatly on physicochemical properties of the peptides. Our designed peptides having the Arg-DHis-Arg template and two lipidic moieties facilitate high yield of intracellular delivery of siRNA. Additionally, unsaturated lipid, linoleic acid moieties were introduced to promote fusogenicity and facilitate endosomal release and cytosolic delivery. Interestingly, such protease-resistant peptides provide serum stability to siRNA and exhibit high efficacy of erk1 and erk2 gene silencing in the triple negative breast cancer (TNBC) cell line. The peptide having two linoleyl moieties demonstrated comparable efficacy with commercial transfection reagent HiPerFect, as evidenced by the erk1 and erk2 gene knockdown experiment. Additionally, our study shows that ERK1/2 silencing siRNA and doxorubicin-loaded gramicidin-mediated combination therapy is more effective than siRNA-mediated gene silencing-based monotherapy for TNBC treatment.