Background: Pah1p is a phosphatidic acid phosphatase that generates diacylglycerol. Results: Deletion of PAH1 alters the sorting of fusion factors to the vacuole and inhibits fusion. Conclusion: Conversion of phosphatidic acid to diacylglycerol is integral to vacuole homeostasis. Significance: This is the first report that links a Lipin 1 homologue to the recruitment and activation of a Rab GTPase or the sorting of SNARE proteins.
Summary
We present strategies for chemical shift assignments of large proteins by magic-angle spinning (MAS) solid-state NMR (SSNMR), using the 21-kDa disulfide bond forming enzyme DsbA as a prototype. Previous studies have demonstrated that complete de novo assignments are possible for proteins up to ~17 kDa, and partial assignments have been performed for several larger proteins. Here we show that combinations of isotopic labeling strategies, high field correlation spectroscopy and 3D and 4D backbone correlation experiments yield highly confident assignments for more than 90% of the backbone resonances in DsbA. Samples were prepared as nanocrystalline precipitates by a dialysis procedure, resulting in heterogeneous linewidths under 0.2 ppm. Thus, high magnetic fields, selective decoupling pulse sequences, and sparse isotopic labeling all improved spectral resolution. Assignments by amino acid type were facilitated by particular combinations of pulse sequences and isotopic labeling; for example, TEDOR experiments enhanced sensitivity for Pro and Gly residues, 2-13C-glycerol labeling clarified Val, Ile and Leu assignments, IPAP correlation spectra enabled interpretation of otherwise crowded Glx/Asx sidechain regions, and 3D NCACX experiments on 2-13C-glycerol samples provided unique sets of aromatic (Phe, Tyr, Trp) correlations. Together with high sensitivity CANCOCA 4D and CANCOCX 3D experiments, unambiguous backbone walks could be performed throughout the majority of the sequence. At 189 residues, DsbA represents the largest monomeric unit for which essentially complete solid-state NMR assignments have so far been achieved. These results will facilitate studies of nanocrystalline DsbA structure and dynamics and enable analysis of its 41-kDa covalent complex with the membrane protein DsbB, for which we demonstrate a high-resolution 2D 13C-13C spectrum.
Ybt1p is a class C ABC transporter (ATP-binding cassette transporter) that is localized to the vacuole of Saccharomyces cerevisiae. Although Ybt1p was originally identified as a bile acid transporter, it has also been found to function in other capacities, including the translocation of phosphatidylcholine to the vacuole lumen, and the regulation of Ca2+ homoeostasis. In the present study we found that deletion of YBT1 enhanced in vitro homotypic vacuole fusion by up to 50 % relative to wild-type vacuoles. The increased vacuole fusion was not due to aberrant protein sorting of SNAREs (soluble N-ethylmaleimide-sensitive factor-attachment protein receptors) or recruitment of factors from the cytosol such as Ypt7p and the HOPS (homotypic fusion and vacuole protein sorting) tethering complex. In addition, ybt1Δ vacuoles displayed no observable differences in the formation of SNARE complexes, interactions between SNAREs and HOPS, or formation of vertex microdomains. However, the absence of Ybt1p caused significant changes in Ca2+ transport during fusion. One difference was the prolonged Ca2+ influx exhibited by ybt1Δ vacuoles at the start of the fusion reaction. We also observed a striking delay in SNARE-dependent Ca2+ efflux. As vacuole fusion can be inhibited by high Ca2+ concentrations, we suggest that the delayed efflux in ybt1Δ vacuoles leads to the enhanced SNARE function.
SummaryHomotypic vacuole fusion requires SNAREs, the Rab Ypt7p, the tethering complex HOPS, regulatory lipids and actin. In Saccharomyces cerevisiae, actin functions at two stages of vacuole fusion. Pre-existing actin filaments are depolymerized to allow docking and assembly of the vertex ring (a microdomain enriched in proteins and lipids that mediate fusion). Actin is then polymerized late in the pathway to aid fusion. Here, we report that the fusion machinery regulates the accumulation of actin at the vertex ring. Using Cy3-labeled yeast actin to track its dynamics, we found that its vertex enrichment was abolished when actin monomers were stabilized by latrunculin-B, independent of the extent of incorporation. By contrast, stabilization of filamentous actin with jasplakinolide markedly augmented actin vertex enrichment. Importantly, agents that inhibit SNAREs, Ypt7p and HOPS inhibited the vertex enrichment of actin, demonstrating that the cytoskeleton and the fusion machinery are interdependently regulated. Actin mobilization was also inhibited by ligating ergosterol and PtdIns(3)P, whereas the ligation or modification of PtdIns(4,5)P 2 augmented the vertex enrichment of actin. The proteins and lipids that regulated actin mobilization to the vertex did not affect the total incorporation of Cy3-actin, indicating that actin mobilization and polymerization activities can be dissociated during membrane fusion.
Background: Ycf1p is a ABCC transporter that is localized to the vacuole and that was initially characterized as a cadmium transporter. Results: Deletion of YCF1 inhibits vacuole fusion in part by excluding the soluble SNARE Vam7p.
Conclusion:The vacuole fusion machinery requires Ycf1p function for efficient fusion. Significance: This is the first report that an ABCC protein affects fusion through the recruitment of a SNARE.
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