In order to undertake in plant cell the study of the endoplasmic reticulum (ER)-Golgi apparatus (GA) protein and/or lipid vesicular transport pathway, expressed sequence tag (EST) coding for a homologue to the yeast soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) Ykt6p has been cloned in Arabidopsis thaliana by reverse transcription polymerase chain reaction (RT-PCR). The corresponding protein was over-expressed as a recombinant histidine-tag (his-tag) protein in E. coli. Starting from one litter of culture, an ultrasonic homogenization was performed for cell disruption and after centrifugation the Arabidopsis Ykt6p SNARE present in inclusion bodies in the pellet was solubilized. After centrifugation, the clarified feedstock obtained was injected onto an immobilized metal affinity chromatography (IMAC) in presence of 6 M guanidine and on-column refolding was performed. Folded and subsequently purified (94% purity) recombinant protein was obtained with 82% of recovery.
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Analysis of interactions between SNARE proteins using imaging ellipsometer coupled with microfluidic array.
Sci Rep
June 2014
Institute of Biophysics, Chinese Academy of Sciences, #15, Datun Rd., Beijing, 100101, China.
The soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins are small and abundant membrane-bound proteins, whose specific interactions mediate membrane fusion during cell fusion or cellular trafficking. In this study, we report the use of a label-free method, called imaging ellipsometer to analyze the interactions among three SNAREs, namely Sec22p, Ykt6p and Sso2p. The SNAREs were immobilized on the silicon wafer and then analyzed in a pairwise mode with microfluidic array, leading us to discover the interactions between Ykt6p and Sso2p, Sec22p and Sso2p.
View Article and Find Full Text PDFStimulation of actin polymerization by vacuoles via Cdc42p-dependent signaling.
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Department of Cell Biology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
We have previously shown that actin ligands inhibit the fusion of yeast vacuoles in vitro, which suggests that actin remodeling is a subreaction of membrane fusion. Here, we demonstrate the presence of vacuole-associated actin polymerization activity, and its dependence on Cdc42p and Vrp1p. Using a sensitive in vitro pyrene-actin polymerization assay, we found that vacuole membranes stimulated polymerization, and this activity increased when vacuoles were preincubated under conditions that support membrane fusion.
View Article and Find Full Text PDFIdentification of the yeast R-SNARE Nyv1p as a novel longin domain-containing protein.
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Department of Biochemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, Special Administrative Region of China.
Using nuclear magnetic resonance spectroscopy, we establish that the N-terminal domain of the yeast vacuolar R-SNARE Nyv1p adopts a longin-like fold similar to those of Sec22b and Ykt6p. Nyv1p is sorted to the limiting membrane of the vacuole via the adaptor protein (AP)3 adaptin pathway, and we show that its longin domain is sufficient to direct transport to this location. In contrast, we found that the longin domains of Sec22p and Ykt6p were not sufficient to direct their localization.
View Article and Find Full Text PDFIdentification of functionally interacting SNAREs by using complementary substitutions in the conserved '0' layer.
Mol Biol Cell
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Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes form bundles of four parallel alpha-helices. The central '0' layer of interacting amino acid side chains is highly conserved and contains one arginine and three glutamines, leading to the classification of SNAREs into R, Qa, Qb, and Qc-SNAREs. Replacing one of the glutamines with arginine in the yeast exocytotic SNARE complex is either lethal or causes a conditional growth defect that is compensated by replacing the R-SNARE arginine with glutamine.
View Article and Find Full Text PDFIntramolecular protein-protein and protein-lipid interactions control the conformation and subcellular targeting of neuronal Ykt6.
J Cell Sci
September 2004
University of Michigan, Department of Molecular, Cellular and Developmental Biology, Ann Arbor, MI 48109-1048, USA.
Although the membrane-trafficking functions of most SNAREs are conserved from yeast to humans, some mammalian SNAREs have evolved specialized functions unique to multicellular life. The mammalian homolog of the prenylated yeast SNARE Ykt6p might be one such example, because rat Ykt6 is highly expressed only in brain neurons. Furthermore, neuronal Ykt6 displayed a remarkably specialized, punctate localization that did not overlap appreciably with conventional compartments of the endomembrane system, suggesting that Ykt6 might be involved in a pathway unique to or specifically modified for neuronal function.
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