Leonora F. Ciufo

Elongation arrest is a physiologically important function of signal recognition particle

Signal recognition particle (SRP) targets proteins for co‐translational insertion through or into the endoplasmic reticulum membrane. Mammalian SRP slows nascent chain elongation by the ribosome during targeting in vitro. This ‘elongation arrest’ activity requires the SRP9/14 subunit of the particle and interactions of the C‐terminus of SRP14. We have purified SRP from Saccharomyces cerevisiae and demonstrated that it too has elongation arrest activity. A yeast SRP containing Srp14p truncated at its C‐terminus (ΔC29) did not maintain elongation arrest, was substantially deficient in promoting translocation and interfered with targeting by wild‐type SRP. In vivo, this mutation conferred a constitutive defect in the coupling of protein translation and translocation and temperature‐sensitive growth, but only a slight defect in protein translocation. In combination, these data indicate that the primary defect in SRP ΔC29 is in elongation arrest, and that this is a physiologically important and conserved function of eukaryotic SRP.

Nuclear export of yeast signal recognition particle lacking Srp54p by the Xpo1p/Crm1p NES-dependent pathway

Abstract Background: The movement of macromolecules through the nuclear pores requires energy and transport receptors that bind both cargo and nuclear pores. Different molecules/complexes often require different transport receptors. The signal recognition particle (SRP) is a conserved cytosolic ribonucleoprotein that targets proteins to the endoplasmic reticulum. Previous studies have shown that the export of SRP RNA from the nucleus requires trans -acting factors and that SRP may be at least partly assembled in the nucleus, but little else is known about how it is assembled and exported into the cytoplasm. Results: Of the six proteins that constitute the yeast SRP, we found that all except Srp54p were imported into the nucleus. Four of these had nucleolar pools. The same four proteins are required for stability of the yeast SRP RNA scR1, suggesting that they assemble with the RNA in the nucleus to form a central core SRP. This core SRP was a competent export substrate. Of the remaining components, Sec65p entered the nucleus and was assembled onto the core particle there, whereas Srp54p was solely cytoplasmic. The export of SRP from the nucleus required the transport receptor Xpo1p/Crm1p and Yrb2p, both components of the pathway that exports leucine-rich nuclear export signal (NES)-containing proteins from the nucleus. Conclusions: The SRP is assembled in the nucleus into a complex lacking only Srp54p. It is then exported through the NES pathway into the cytoplasm where Srp54p binds to it. This transport route for a ribonucleoprotein complex is so far unique in yeast.

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.

A Random Mutagenesis Approach to Isolate Dominant-Negative Yeast sec1 Mutants Reveals a Functional Role for Domain 3a in Yeast and Mammalian Sec1/Munc18 Proteins

SNAP receptor (SNARE) and Sec1/Munc18 (SM) proteins are required for all intracellular membrane fusion events. SNAREs are widely believed to drive the fusion process, but the function of SM proteins remains unclear. To shed light on this, we screened for dominant-negative mutants of yeast Sec1 by random mutagenesis of a GAL1-regulated SEC1 plasmid. Mutants were identified on the basis of galactose-inducible growth arrest and inhibition of invertase secretion. This effect of dominant-negative sec1 was suppressed by overexpression of the vesicle (v)-SNAREs, Snc1 and Snc2, but not the target (t)-SNAREs, Sec9 and Sso2. The mutations isolated in Sec1 clustered in a hotspot within domain 3a, with F361 mutated in four different mutants. To test if this region was generally involved in SM protein function, the F361-equivalent residue in mammalian Munc18-1 (Y337) was mutated. Overexpression of the Munc18-1 Y337L mutant in bovine chromaffin cells inhibited the release kinetics of individual exocytosis events. The Y337L mutation impaired binding of Munc18-1 to the neuronal SNARE complex, but did not affect its binary interaction with syntaxin1a. Taken together, these data suggest that domain 3a of SM proteins has a functionally important role in membrane fusion. Furthermore, this approach of screening for dominant-negative mutants in yeast may be useful for other conserved proteins, to identify functionally important domains in their mammalian homologs.

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. Conceptual translation of the cDNA, that encodes a 58.9 kDa protein, and database searching, revealed that the enzyme belongs to an FAD-dependent oxidoreductase family. Western blotting revealed reductase protein expression exclusively in the microsomal subcellular fraction and primarily in the gut. The protein is peripherally associated with microsomal membranes. 2D-native gel and PAGE analysis revealed that the reductase is part of a large complex with molecular weight approximately 250kDa. The protein occurs in midgut microsomes at a fairly constant level throughout development in the last two instars, but is drastically reduced during the wandering stage in preparation for metamorphosis. Putative Broad Complex transcription factor-binding sites detectable upstream of the DHCR24 gene may play a role in this down-regulation.