Enzyme-coupled assays for flip-flop of acyl-Coenzyme A in liposomes

Acyl-Coenzyme A is made in the cytosol. Certain enzymes using acyl-CoA seem to operate in the lumen of the ER but no corresponding flippases for acyl-CoA or an activated acyl have been described. In order to test the ability of purified candidate flippases to operate the transport of acyl-CoA through lipid bilayers in vitro we developed three enzyme-coupled assays using large unilamellar vesicles (LUVs) obtained by detergent removal. The first assay uses liposomes encapsulating a water-soluble acyl-CoA:glycerol-3-phosphate acyl transferase plus glycerol-3-phosphate (G3P). It measures formation of [3H]lyso-phosphatidic acid inside liposomes after [3H]palmitoyl-CoA has been added from outside. Two other tests use empty liposomes containing [3H]palmitoyl-CoA in the inner membrane leaflet, to which either soluble acyl-CoA:glycerol-3-phosphate acyl transferase plus glycerol-3-phosphate or alkaline phosphatase are added from outside. Here one can follow the appearance of [3H]lyso-phosphatidic acid or of dephosphorylated [3H]acyl-CoA, respectively, both being made outside the liposomes. Although the liposomes may retain small amounts of detergent, all these tests show that palmitoyl-CoA crosses the lipid bilayer only very slowly and that the lipid composition of liposomes barely affects the flip-flop rate. Thus, palmitoyl-CoA cannot cross the membrane spontaneously implying that in vivo some transport mechanism is required

Archaeological finds from Križna jama cave

V prispevku obravnavamo arheološke najdbe iz spele-ološko pomembne Križne jame na Notranjskem, ki je od leta 1955 odprta tudi za turistične obiske.V  znanstveni  literaturi  je  jama  od  konca  19.  stoletja znana  predvsem  po  paleontoloških  ostankih  jamskega medveda  (Ursus  spelaeus).  Kot  arheološko  najdišče  pa  je bila  zabeležena  šele  leta  1973,  vendar  je  širši  strokovni javnosti ostala do danes neznana.Antropogenih struktur v jami še ne poznamo. Izjema je morda  plato  1  v  vhodnem  delu  jame,  kjer  so  raziskovalci naleteli na drobce oglja in fragmente keramike.Najstarejše  arheološke  najdbe  lahko  sodijo  v  bakreno dobo,  kronološko  bolj  zanesljive  pa  uvrščamo  okvirno  v srednjo bronasto dobo, kamor moremo datirati tudi prve obiske človeka v jami. Posamične najdbe in napisi na jamskih stenah kažejo, da je človek jamo obiskoval zaradi različnih razlogov tudi v poznejših obdobjih, vse do danes.This paper discusses the archaeological finds from the speleologically important Križna jama cave, located in the Notranjska region. Since 1955, the cave has been open for tourist visits.From  the  end  of  the  19th  century,  the  cave  had  been known  in  scientific  circles  mostly  because  of  the  pale-ontological  remains  of  cave  bear  (Ursus  spelaeus).  It  was only  as  late  as  1973  when  the  cave  was  recognised  as  an archaeological site; however, it has remained unknown to the wider professional public until the present.No anthropogenic structures have been discovered in the cave. One exception is perhaps plateau 1, at the entrance of the cave, where researchers came across some charcoal flecks and pottery fragments.The  earliest  archaeological  finds  can  be  dated  to  the C opper  Age.  However,  chronologically  reliable  finds originate  broadly  from  the  Middle  Bronze  Age,  which  is also  the  period  of  the  earliest  human  visits  to  the  cave. Individual finds and inscriptions on the walls of the cave indicate that people have been, for one reason or another, visiting the cave ever since

A neurotoxic phospholipase A2 impairs yeast amphiphysin activity and reduces endocytosis.

BACKGROUND: Presynaptically neurotoxic phospholipases A(2) inhibit synaptic vesicle recycling through endocytosis. PRINCIPAL FINDINGS: Here we provide insight into the action of a presynaptically neurotoxic phospholipase A(2) ammodytoxin A (AtxA) on clathrin-dependent endocytosis in budding yeast. AtxA caused changes in the dynamics of vesicle formation and scission from the plasma membrane in a phospholipase activity dependent manner. Our data, based on synthetic dosage lethality screen and the analysis of the dynamics of sites of endocytosis, indicate that AtxA impairs the activity of amphiphysin. CONCLUSIONS: We identified amphiphysin and endocytosis as the target of AtxA intracellular activity. We propose that AtxA reduces endocytosis following a mechanism of action which includes both a specific protein-protein interaction and enzymatic activity, and which is applicable to yeast and mammalian cells. Knowing how neurotoxic phospholipases A(2) work can open new ways to regulate endocytosis

The heptad repeat domain 1 of Mitofusin has membrane destabilization function in mitochondrial fusion

International audienceMitochondria are double-membrane-bound organelles that constantly change shape through membrane fusion and fission. Outer mitochondrial membrane fusion is controlled by Mitofusin, whose molecular architecture consists of an N-terminal GTPase domain, a first heptad repeat domain (HR1), two transmembrane domains, and a second heptad repeat domain (HR2). The mode of action of Mitofusin and the specific roles played by each of these functional domains in mitochondrial fusion are not fully understood. Here, using a combination of in situ and in vitro fusion assays, we show that HR1 induces membrane fusion and possesses a conserved amphipathic helix that folds upon interaction with the lipid bilayer surface. Our results strongly suggest that HR1 facilitates membrane fusion by destabilizing the lipid bilayer structure, notably in membrane regions presenting lipid packing defects. This mechanism for fusion is thus distinct from that described for the heptad repeat domains of SNARE and viral proteins, which assemble as membrane-bridging complexes, triggering close membrane apposition and fusion, and is more closely related to that of the C-terminal amphipathic tail of the Atlastin protein

AtxA inhibits endocytosis in an Rvs161-dependent manner.

<p>Relative uptake rates of LY in AtxA-expressing and control strains with wild-type and different <i>rvs</i> deletion backgrounds. The bars represent standard deviations of at least three independent measurements. AtxA-expressing strains were compared to the corresponding control strains, and p-values were calculated using the <i>t-</i>test at a 95% confidence interval. Statistically significant differences are denoted with a star (*).</p

Binding of yeast 14-3-3 proteins to phospholipid vesicles.

<p>Surface plasmon resonance was used to analyze binding of 14-3-3 proteins to phospholipid vesicles. The response after 180 seconds of dissociation phase is shown. Av. – average response in response units (RU), s.d. – standard deviation in RU of at least three independent measurements.</p

Rvs proteins are needed for the effect of AtxA on endocytosis.

<p><b>A</b> Average Sla1-GFP patch lifetimes for different <i>rvs</i> deletion strains and <i>yap1802Δ</i> deletion strain expressing AtxA from a single copy integrated in the genome, and corresponding control strains. <b>B</b> Localization of Sla1-GFP in <i>rvs161Δ</i>, <i>rvs167Δ</i>, the <i>rvs161Δ rvs167Δ</i> double deletion strain and <i>yap1802Δ</i>. <b>C</b> Effect of additional expression of either <i>RVS161</i> or <i>RVS167</i> on average Sla1-GFP patch lifetimes for WT and <i>rvs</i> deletion strains expressing AtxA from a single copy integrated in the genome and corresponding control strains. <b>D</b> Average Sla1-GFP patch lifetimes for the wild-type and <i>bmh1Δ</i> and <i>bmh2Δ</i> deletion strains expressing AtxA from plasmid, and corresponding control strains. <b>E</b> Average lifetimes of Rvs161-GFP (left) and Rvs167-GFP (right) patches for the wild-type (ctrl) and AtxA-expressing cells. <b>F</b> Localization of Rvs161-GFP and Rvs167-GFP in AtxA-expressing and control cells. The bars on graphs mark the standard deviation. To determine the average patch lifetimes at least 100 patches from several cells were analyzed. Sla1-GFP movies were taken with a 1 frame/second and Rvs161-GFP and Rvs167-GFP movies with a 4 frames/second interval. Scale bar on the micrographs is 4 µm. AtxA-expressing strains were compared to the corresponding control strains, and p-values were calculated using the <i>t-</i>test at a 95% confidence interval. Statistically significant differences are denoted with a star (*).</p