It was chosen to be large enough to produce reproducible human gland stimulation mediated by increasedi in control airwaysbut

When expressed in yeast, LegC7 disrupts biosynthetic vacuole-directed cargo that emanate from the Golgi, such as CPS and Sna3p. In both cases, the predominant phenotype consists of numerous punctate structures that localize to the cell periphery. Because these proteins are trafficked via similar mechanisms, we suspect that both GFP-CPS and Sna3-GFP are accumulating in the same physiological compartments; perhaps early endosomes that are unable to either mature or fuse to downstream compartments. In addition, by following fluid-phase endocytosis with the soluble dye Lucifer Yellow, we find that yeast cells expressing LegC7 accumulate this marker within the cytosol. Therefore, LegC7 does not completely prevent endocytosis, as the dye is still able to enter the cell, but the LY-containing endosomes fail to deliver their cargo to the vacuole. Interestingly, strains deleted for vps21 and ypt52, the major Rab-family GTPases of the early SAR131675 abmole bioscience endocytic pathway, are also reported to display a similar LY accumulation phenotype ; this phenotypic similarity to LegC7-expressing strains further suggests that LegC7 may be capable of modulating the early endocytic pathway. It is unknown, however, whether LegC7 directly manipulates these Rab GTPases or the fusion events they catalyze, and therefore requires further study. Upon LegC7 expression, the fluorescent styryl dye, FM4-64 was not seen to accumulate in yeast vacuole membranes, but instead was contained within punctate structures around the cell periphery. The localization pattern observed with FM4-64 mirrors the aberrant accumulation of GFP-CPS and Sna3-GFP in LegC7- expressing cells, leading us to hypothesize that these structures represent the same physiological compartment. Using a GFP tagged version of the a-Factor, Ste3p, we determined that LegC7 also prevented proper vacuolar delivery of receptor mediated endocytic cargoes yet did not disrupt the actual endocytic event. Our data indicates that LegC7 manipulates traffic involving endosomal maturation, however we wondered if the effects of LegC7 were specific to the endosomal system, or rather represented a global disruption of traffic. As the yeast vacuole receives cargo from at least two other pathways we sought to determine if LegC7 disrupted these pathways as well. In order the probe the ALP pathway which moves cargo directly from the late Golgi to the vacuole in an AP-3 dependent manner we utilized GFP-Vam3, a well-characterized vacuolar SNARE that is known to traffic through the ALP pathway. Localization of GFP-Vam3 was not disrupted by LegC7 expression, nor was the processing of a Cvt-delivered protein, Ape1p. These data indicate that LegC7 specifically disrupts cargo that is required to traffic through endosomes to vacuoles, while not disrupting global cellular trafficking events. Mutation of the asparagine 242 of LegC7 to either isoleucine or leucine results in a nontoxic derivative of LegC7 that also lacks the endocytic disruption phenotypes of wild type LegC7. Based on in silico calculations, this residue is predicted to fall in the very beginning of the putative second coiled-coil domain of LegC7. Furthermore, models predict that replacing this residue with a large aliphatic amino acid alters the predicted linking region between coiled coil region 1 and 2, suggesting that the presence of this domain is critical for proper LegC7 folding or function in vivo. In support of these data, early studies with LegC7 found that large deletions of this central coiled-coil domain produced a non-toxic protein, and that the C-terminal coiled-coil domain of LegC7 was not important for toxicity. As the elimination of LegC7 toxicity in our study is fairly specific, we suspect that significant structural changes are induced in LegC7N242I, but will not be fully appreciated until crystallographic data are obtained. Recently, the N-terminal portion of a related Legionella effector protein, LegC3, was crystallized, resulting in a structure that did not share close homology with any currently known structure. As this crystal structure did not match in silico predictions, the structure of LegC7 may provide a new role for the N242 residue in LegC7 function. Finally, we find that deletions of VPS27, and ESCRT-0 complex member, partially reversed the toxic effects of LEGC7 expression. This effect is not the result of mislocalization of LegC7, but could be explained by the reduction of LegC7 levels through enhanced proteolytic turnover or reduced LegC7 expression in backgrounds; direct interactions between Vps27p and LegC7 in vitro were not detected.

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