scholarly journals SNARE-catalyzed Fusion Events Are Regulated by Syntaxin1A–Lipid Interactions

2008 ◽  
Vol 19 (2) ◽  
pp. 485-497 ◽  
Author(s):  
Alice D. Lam ◽  
Petra Tryoen-Toth ◽  
Bill Tsai ◽  
Nicolas Vitale ◽  
Edward L. Stuenkel

Membrane fusion is a process that intimately involves both proteins and lipids. Although the SNARE proteins, which ultimately overcome the energy barrier for fusion, have been extensively studied, regulation of the energy barrier itself, determined by specific membrane lipids, has been largely overlooked. Our findings reveal a novel function for SNARE proteins in reducing the energy barrier for fusion, by directly binding and sequestering fusogenic lipids to sites of fusion. We demonstrate a specific interaction between Syntaxin1A and the fusogenic lipid phosphatidic acid, in addition to multiple polyphosphoinositide lipids, and define a polybasic juxtamembrane region within Syntaxin1A as its lipid-binding domain. In PC-12 cells, Syntaxin1A mutations that progressively reduced lipid binding resulted in a progressive reduction in evoked secretion. Moreover, amperometric analysis of fusion events driven by a lipid-binding–deficient Syntaxin1A mutant (5RK/A) demonstrated alterations in fusion pore dynamics, suggestive of an energetic defect in secretion. Overexpression of the phosphatidic acid–generating enzyme, phospholipase D1, completely rescued the secretory defect seen with the 5RK/A mutant. Moreover, knockdown of phospholipase D1 activity drastically reduced control secretion, while leaving 5RK/A-mediated secretion relatively unaffected. Altogether, these data suggest that Syntaxin1A–lipid interactions are a critical determinant of the energetics of SNARE-catalyzed fusion events.

2014 ◽  
Vol 106 (2) ◽  
pp. 716a
Author(s):  
Priya Putta ◽  
Johanna M. Rankenberg ◽  
Christa Testerink ◽  
Edgar E. Kooijman

2017 ◽  
Vol 13 ◽  
pp. 9-17
Author(s):  
Magda Piaścik ◽  
Jolanta Zegarlińska ◽  
Aleksander F. Sikorski ◽  
Aleksander Czogalla

Flotation is one of the best method for preliminary identification of protein-lipid interactions. In most widely used approach it utilizes large unilamellar vesicles, that are excellent models of freestanding membranes and do not require any additional components, like solid supports or beads that are needed in other methods commonly used for protein-lipid binding studies. Here we present results obtained during our studies on phosphatidic acid - syndapin interactions and discuss some technical aspects of this method underlying how relatively small changes in the conditions can influence the results.


1993 ◽  
Vol 268 (6) ◽  
pp. 4037-4042
Author(s):  
R. van't Hof ◽  
W. van Klompenburg ◽  
M. Pilon ◽  
A. Kozubek ◽  
G. de Korte-Kool ◽  
...  

Biomedicines ◽  
2021 ◽  
Vol 9 (1) ◽  
pp. 45
Author(s):  
Suresh Velnati ◽  
Sara Centonze ◽  
Federico Girivetto ◽  
Daniela Capello ◽  
Ricardo M. Biondi ◽  
...  

PKCζ and PKCι/λ form the atypical protein kinase C subgroup, characterised by a lack of regulation by calcium and the neutral lipid diacylglycerol. To better understand the regulation of these kinases, we systematically explored their interactions with various purified phospholipids using the lipid overlay assays, followed by kinase activity assays to evaluate the lipid effects on their enzymatic activity. We observed that both PKCζ and PKCι interact with phosphatidic acid and phosphatidylserine. Conversely, PKCι is unique in binding also to phosphatidylinositol-monophosphates (e.g., phosphatidylinositol 3-phosphate, 4-phosphate, and 5-phosphate). Moreover, we observed that phosphatidylinositol 4-phosphate specifically activates PKCι, while both isoforms are responsive to phosphatidic acid and phosphatidylserine. Overall, our results suggest that atypical Protein kinase C (PKC) localisation and activity are regulated by membrane lipids distinct from those involved in conventional PKCs and unveil a specific regulation of PKCι by phosphatidylinositol-monophosphates.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alessandra Luchini ◽  
Samantha Micciulla ◽  
Giacomo Corucci ◽  
Krishna Chaithanya Batchu ◽  
Andreas Santamaria ◽  
...  

AbstractSARS-CoV-2 spike proteins are responsible for the membrane fusion event, which allows the virus to enter the host cell and cause infection. This process starts with the binding of the spike extramembrane domain to the angiotensin-converting enzyme 2 (ACE2), a membrane receptor highly abundant in the lungs. In this study, the extramembrane domain of SARS-CoV-2 Spike (sSpike) was injected on model membranes formed by supported lipid bilayers in presence and absence of the soluble part of receptor ACE2 (sACE2), and the structural features were studied at sub-nanometer level by neutron reflection. In all cases the presence of the protein produced a remarkable degradation of the lipid bilayer. Indeed, both for membranes from synthetic and natural lipids, a significant reduction of the surface coverage was observed. Quartz crystal microbalance measurements showed that lipid extraction starts immediately after sSpike protein injection. All measurements indicate that the presence of proteins induces the removal of membrane lipids, both in the presence and in the absence of ACE2, suggesting that sSpike molecules strongly associate with lipids, and strip them away from the bilayer, via a non-specific interaction. A cooperative effect of sACE2 and sSpike on lipid extraction was also observed.


2021 ◽  
Author(s):  
Xiao Xiao Zhang ◽  
John William Young ◽  
Leonard J Foster ◽  
Franck Duong

Many soluble proteins interact with membranes to perform important biological functions, including signal transduction, regulation, transport, trafficking and biogenesis. Despite their importance, these protein-membrane interactions are difficult to characterize due to their often-transient nature as well as phospholipids' poor solubility in aqueous solution. Here, we employ nanodiscs - small, water-soluble patches of lipid bilayer encircled with amphipathic scaffold proteins - along with quantitative proteomics to identify lipid-binding proteins in S. cerevisiae. Using nanodiscs reconstituted with yeast total lipid extracts or only phosphatidylethanolamine (PE-nanodiscs), we capture several known membrane-interacting proteins, including the Rab GTPases Sec4 and Ypt1, which play key roles in vesicle trafficking. Utilizing PE-nanodiscs enriched with phosphatidic acid (PEPA-nanodiscs), we specifically capture a member of the Hsp40/J-protein family, Caj1, whose function has recently been linked to membrane protein quality control. We show that Caj1 interaction with liposomes containing PA is modulated by pH and PE lipids, and depends on two patches of positively charged residues near the C-terminus of the protein. The protein Caj1 is the first example of an Hsp40/J-domain protein with affinity for membranes and phosphatidic acid lipid specificity. These findings highlight the utility of the nanodisc system to identify and characterize protein-lipid interactions that may not be evident using other methods.


Toxins ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 226
Author(s):  
Sudarat Tharad ◽  
Boonhiang Promdonkoy ◽  
José L. Toca-Herrera

Cytolytic toxin (Cyt) is a toxin among Bacillus thuringiensis insecticidal proteins. Cyt toxin directly interacts with membrane lipids for cytolytic action. However, low hemolytic activity is desired to avoid non-specific effects in mammals. In this work, the interaction between Cyt2Aa2 toxin and model lipid bilayers mimicking the erythrocyte membrane was investigated for Cyt2Aa2 wild type (WT) and the T144A mutant, a variant with lower hemolytic activity. Quartz crystal microbalance with dissipation (QCM-D) results revealed a smaller lipid binding capacity for the T144A mutant than for the WT. In particular, the T144A mutant was unable to bind to the phosphatidylcholine lipid (POPC) bilayer. However, the addition of cholesterol (Chol) or sphingomyelin (SM) to the POPC bilayer promoted binding of the T144 mutant. Moreover, atomic force microscopy (AFM) images unveiled small aggregates of the T144A mutant on the 1:1 sphingomyelin/POPC bilayers. In contrast, the lipid binding trend for WT and T144A mutant was comparable for the 1:0.4 POPC/cholesterol and the 1:1:1 sphingomyelin/POPC/cholesterol bilayers. Furthermore, the binding of WT and T144A mutant onto erythrocyte cells was investigated. The experiments showed that the T144A mutant and the WT bind onto different areas of the erythrocyte membrane. Overall the results suggest that the T144 residue plays an important role for lipid binding.


ChemInform ◽  
2003 ◽  
Vol 34 (28) ◽  
Author(s):  
N. T. Ktistakis ◽  
C. Delon ◽  
M. Manifava ◽  
E. Wood ◽  
I. Ganley ◽  
...  

Endocrinology ◽  
2013 ◽  
Vol 154 (3) ◽  
pp. 1235-1246 ◽  
Author(s):  
Matjaž Stenovec ◽  
Paula P. Gonçalves ◽  
Robert Zorec

Abstract In this study we used live-cell immunocytochemistry and confocal microscopy to study the release from a single vesicle in a simplified system called membrane lawns. The lawns were prepared by exposing differentiated pituitary prolactin (PRL)-secreting cells to a hypoosmotic shear stress. The density of the immunolabeled ternary soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) complexes that bind complexin was approximately 10 times lower than the PRL-positive, lawn-resident vesicles; this indicates that some but not all vesicles are associated with ternary SNARE complexes. However, lawn-resident PRL vesicles colocalized relatively well with particular SNARE proteins: synaptobrevin 2 (35%), syntaxin 1 (22%), and 25-kDa synaptosome associated protein (6%). To study vesicle discharge, we prepared lawn-resident vesicles, derived from atrial natriuretic peptide tagged with emerald fluorescent protein (ANP.emd)-transfected cells, which label vesicles. These maintained the structural passage to the exterior because approximately 40% of ANP.emd-loaded vesicles were labeled by extracellular PRL antibodies. Cargo release from the lawn-resident vesicles, monitored by the decline in the ANP.emd fluorescence intensity, was similar to that in intact cells. It is likely that SNARE proteins are required for calcium-dependent release from these vesicles. This is because the expression of the dominant-negative SNARE peptide, which interferes with SNARE complex formation, reduced the number of PRL-positive spots per cell (PRL antibodies placed extracellularly) significantly, from 58 ± 9 to 4 ± 2. In dominant-negative SNARE-treated cells, the PRL-positive area was reduced from 0.259 ± 0.013 to 0.123 ± 0.014 μm2, which is consistent with a hindered vesicle luminal access for extracellular PRL antibodies. These results indicate that vesicle discharge is regulated by SNARE-mediated fusion pore widening.


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