The Sec1p/Munc18 protein Vps45p binds its cognate SNARE proteins via two distinct modes

Sec1p/Munc18 (SM) proteins are essential for SNARE-mediated membrane trafficking. The formulation of unifying hypotheses for the function of the SM protein family has been hampered by the observation that two of its members bind their cognate syntaxins (Sxs) in strikingly different ways. The SM protein Vps45p binds its Sx Tlg2p in a manner analogous to that captured by the Sly1p–Sed5p crystal structure, whereby the NH2-terminal peptide of the Sx inserts into a hydrophobic pocket on the outer face of domain I of the SM protein. In this study, we report that although this mode of interaction is critical for the binding of Vps45p to Tlg2p, the SM protein also binds Tlg2p-containing SNARE complexes via a second mode that involves neither the NH2 terminus of Tlg2p nor the region of Vps45p that facilitates this interaction. Our findings point to the possibility that SM proteins interact with their cognate SNARE proteins through distinct mechanisms at different stages in the SNARE assembly/disassembly cycle

Phosphomimetic Mutation of Ser-187 of SNAP-25 Increases both Syntaxin Binding and Highly Ca2+-sensitive Exocytosis

The phosphorylation targets that mediate the enhancement of exocytosis by PKC are unknown. PKC phosporylates the SNARE protein SNAP-25 at Ser-187. We expressed mutants of SNAP-25 using the Semliki Forest Virus system in bovine adrenal chromaffin cells and then directly measured the Ca2+ dependence of exocytosis using photorelease of caged Ca2+ together with patch-clamp capacitance measurements. A flash of UV light used to elevate [Ca2+]i to several μM and release the highly Ca2+-sensitive pool (HCSP) of vesicles was followed by a train of depolarizing pulses to elicit exocytosis from the less Ca2+-sensitive readily releasable pool (RRP) of vesicles. Carbon fiber amperometry confirmed that the amount and kinetics of catecholamine release from individual granules were similar for the two phases of exocytosis. Mimicking PKC phosphorylation with expression of the S187E SNAP-25 mutant resulted in an approximately threefold increase in the HCSP, whereas the response to depolarization increased only 1.5-fold. The phosphomimetic S187D mutation resulted in an ∼1.5-fold increase in the HCSP but a 30% smaller response to depolarization. In vitro binding assays with recombinant SNARE proteins were performed to examine shifts in protein–protein binding that may promote the highly Ca2+-sensitive state. The S187E mutant exhibited increased binding to syntaxin but decreased Ca2+-independent binding to synaptotagmin I. Mimicking phosphorylation of the putative PKA phosphorylation site of SNAP-25 with the T138E mutation decreased binding to both syntaxin and synaptotagmin I in vitro. Expressing the T138E/ S187E double mutant in chromaffin cells demonstrated that enhancing the size of the HCSP correlates with an increase in SNAP-25 binding to syntaxin in vitro, but not with Ca2+-independent binding of SNAP-25 to synaptotagmin I. Our results support the hypothesis that exocytosis triggered by lower Ca2+ concentrations (from the HCSP) occurs by different molecular mechanisms than exocytosis triggered by higher Ca2+ levels

Characterisation of a Desmosterol Reductase Involved in Phytosterol Dealkylation in the Silkworm, Bombyx mori

Most species of invertebrate animals cannot synthesise sterols de novo and many that feed on plants dealkylate phytosterols (mostly C29 and C28) yielding cholesterol (C27). The final step of this dealkylation pathway involves desmosterol reductase (DHCR24)-catalysed reduction of desmosterol to cholesterol. We now report the molecular characterisation in the silkworm, Bombyx mori, of such a desmosterol reductase involved in production of cholesterol from phytosterol, rather than in de novo synthesis of cholesterol. Phylogenomic analysis of putative desmosterol reductases revealed the occurrence of various clades that allowed for the identification of a strong reductase candidate gene in Bombyx mori (BGIBMGA 005735). Following PCR-based cloning of the cDNA (1.6 kb) and its heterologous expression in Saccharomyces cerevisae, the recombinant protein catalysed reduction of desmosterol to cholesterol in an NADH- and FAD- dependent reaction

The C-terminal zinc finger of the catalytic subunit of DNA polymerase δ is responsible for direct interaction with the B-subunit

DNA polymerase δ (Pol δ) plays a central role in eukaryotic chromosomal DNA replication, repair and recombination. In fission yeast, Pol δ is a tetrameric enzyme, comprising the catalytic subunit Pol3 and three smaller subunits, Cdc1, Cdc27 and Cdm1. Previous studies have demonstrated a direct interaction between Pol3 and Cdc1, the B-subunit of the complex. Here it is shown that removal of the tandem zinc finger modules located at the C-terminus of Pol3 by targeted proteolysis renders the Pol3 protein non-functional in vivo, and that the C-terminal zinc finger module ZnF2 is both necessary and sufficient for binding to the B-subunit in vivo and in vitro. Extensive mutagenesis of the ZnF2 module identifies important residues for B-subunit binding. In particular, disruption of the ZnF2 module by substitution of the putative metal-coordinating cysteines with alanine abolishes B-subunit binding and in vivo function. Finally, evidence is presented suggesting that the ZnF region is post-translationally modified in fission yeast cells

DHCR24 activity of <i>B. mori</i> BGIBMGA005735 expressed in <i>S. cerevisiae</i>.

<p>Yeast homogenates containing expressed <i>B. mori</i> BGIBMGA005735 were added to assay mixtures containing desmosterol and various combinations of the cofactors NADPH and FAD. A yeast homogenate containing expressed pYES2 (vector control) was incubated with both cofactors as a negative control. Assays were incubated for 4 h at 37°C and products analysed by GC/MS. MS trace (total ion current) of positive reaction containing cholesterol and negative reaction containing only desmosterol are shown in Fig. 4a. Sterol peaks were calibrated using triplicate reactions containing 5α-cholestane as an internal standard and expressed as a percentage of desmosterol converted into cholesterol compared to the empty vector reaction (Fig 4b). The positions of elution of authentic cholesterol and desmosterol are shown by arrows (Fig. 4a).</p

Western blot analysis of subcellular location and tissue distribution of <i>B. mori</i> DHCR24.

<p>(a) Samples of soluble supernatant (1) and microsomal (2) fractions, normalised for protein concentration were blotted with anti-DHCR24 antibodies. (b) Microsomes produced from various tissue homogenates normalised for protein concentration were probed with anti-DHCR24 antibodies: (1) foregut, (2) midgut, (3) hindgut, (4) testes, (5) ovary, (6) Malpighian tubules, (7) fat body, (8) head.</p

Pathway of transformation of sitosterol into cholesterol.

<p>Pathway of transformation of sitosterol into cholesterol.</p

Membrane association and complex formation of <i>B. mori</i> DHCR24.

<p>(a) Equal aliquots of midgut microsomes were washed with 10 volumes of 5 mM HEPES/NaOH, pH 7.5 on ice for 30 min before reisolating the microsomes and analysing by western blot using anti-DHCR24 antibody. The washes were as follows: (1) HEPES/NaOH, pH 7.5 only, (2) HEPES/NaOH, pH 7.5+200 mM KCl, (3) HEPES/NaOH, pH 7.5+500 mM KCl, (4) 0.1 M Na₂CO_3, pH 11.5. (b) Midgut microsomal protein was solubilised with 10% dodecyl maltoside and separated using native blue gel electrophoresis with native protein markers. This 1st dimension strip was then soaked in β-mercaptoethanol and SDS before running on a standard SDS PAGE gel and analysing by western blot using anti-DHCR24 antibody.</p