The Dynamin-like Protein Vps1 Stimulates Endosome-to-Golgi Fusion in Vitro

Intracellular membrane fusion events can be reconstituted by exploiting isolated organelles from cellular hosts or artificial membranes made of purified phospholipid components. Artificial construction of membranes provides two significant advantages. First, cellular isolation of the endosome-derived vesicles and TGN (trans-Golgi Network) compartments needed for the fusion assay would be extremely challenging. Second, reconstituting the membranes provides the added benefit of controlling size and lipid compositions to functionally mimic the individual membrane architectures and introduce only the purified proteins that are under investigation. For these reasons, I have developed the first simultaneous lipid and content mixing fusion assays that measures the efficacy of endosome-to-TGN fusion and its promoted fusion capability with the dynamin-like protein Vps1 for the purposes of recapitulating the fusion. To quantify lipid mixing between the donor and recipient membrane fluorescent lipids (Rhodamine-PE and NBD-PE) were used, while content mixing was assessed by analyzing the increase of FRET between Cy5- streptavidin and PhycoE-biotin

Integrating Health Literacy Questions into a Statewide Behavioral Risk Factor Surveillance System (BRFSS) Questionnaire

Objectives. The purpose of this pilot study was to evaluate the feasibility of adding health literacy questions to a state health assessment questionnaire. Methods. Researchers conducted a series of telephone interviews (N = 20) to test the telephone administration of three health literacy screening questions with a convenience sample. Feedback obtained during the telephone interviews was used to revise the questions for clarity. The revised questions were proposed as an addition to the Kansas Behavioral Risk Factor Surveillance System (BRFSS). Results. Pilot data included minor modifications to the language of the questions to broaden their interpretation outside of a hospital setting. Most participants (90%, n = 18) had adequate health literacy. The proposed questions were approved for addition to the BRFSS questionnaire. Prompts were added to a telephone script to aid BRFSS survey administrators. Conclusion. As one of the first statewide health literacy assessments, this study has demonstrated one method for collecting baseline data. This new methodology has the potential to impact both patient care and broad public health efforts

Yeast dynamin associates with the GARP tethering complex for endosome-to-Golgi traffic

Yeast dynamin, Vacuolar Protein Sorting 1 (Vps1), has been implicated in recycling traffic from the endosome to the trans-Golgi network (TGN). Previous research showed a genetic interaction of Vps1 with all components of the GARP tethering complex, which anchors vesicles at the late Golgi membrane. We used the yeast two-hybrid system and have identified a 33 amino acid segment of Vps51, a GARP subunit, that interacts with Vps1. Based on sequence homology between Vps51 and its mammalian homolog Ang2 in the 33 amino acids stretch, we identified two key residues of Vps51, E127 and Y129, that bind Vps1. The replacement of these residues led to severe defects in endosome-to-TGN transport of Snc1, providing evidence of the physiological relevance of the interaction of Vps51 with Vps1 for the traffic. Furthermore, our functional analysis revealed that Vps1 acts upstream of Vps51 and that the absence of Vps1 resulted in reduced localization levels of Vps51 and its binding partner Tlg1 to the late Golgi. Taken together, we propose that Vps1 functions with the GARP tethering machinery for efficient tethering/fusion at the TGN

Evaluation of the tert-butyl group as a probe for NMR studies of macromolecular complexes

The development of methyl transverse relaxation optimized spectroscopy has greatly facilitated the study of macromolecular assemblies by solution NMR spectroscopy. However, limited sample solubility and stability has hindered application of this technique to ongoing studies of complexes formed on membranes by the neuronal SNAREs that mediate neurotransmitter release and synaptotagmin-1, the Ca2+ sensor that triggers release. Since the 1H NMR signal of a tBu group attached to a large protein or complex can be observed with high sensitivity if the group retains high mobility, we have explored the use of this strategy to analyze presynaptic complexes involved in neurotransmitter release. For this purpose, we attached tBu groups at single cysteines of fragments of synaptotagmin-1, complexin-1 and the neuronal SNAREs by reaction with 5-(tert-butyldisulfaneyl)-2-nitrobenzoic acid (BDSNB), tBu iodoacetamide or tBu acrylate. The tBu resonances of the tagged proteins were generally sharp and intense, although tBu groups attached with BDSNB had a tendency to exhibit somewhat broader resonances that likely result because of the shorter linkage between the tBu and the tagged cysteine. Incorporation of the tagged proteins into complexes on nanodiscs led to severe broadening of the tBu resonances in some cases. However, sharp tBu resonances could readily be observed for some complexes of more than 200 kDa at low micromolar concentrations. Our results show that tagging of proteins with tBu groups provides a powerful approach to study large biomolecular assemblies of limited stability and/or solubility that may be applicable even at nanomolar concentrations.We thank Ad Bax for the suggestion of exploring the use tBu groups as probes for structural studies of the neurotransmitter release machinery, and Ad Bax, Lewis Kay and Charampalos Kalodimos for fruitful discussions on this subject. The Agilent DD2 console of the 800 MHz spectromenter used for the research presented here was purchased with a shared instrumentation grant from the NIH (S10OD018027 to JR). Rashmi Voleti was supported by a fellowship from the Howard Hughes Medical Institute. The preparation of BDSNB was performed at the NANBIOSIS –CIBER BBN Peptide Synthesis Unit (U3). This work was supported by grant I-1304 from the Welch Foundation (to JR) and by NIH Research Project Award R35 NS097333 (to JR).Peer reviewe