Lipids and the exocytotic machinery of eukaryotic cells

The molecular machines that drive protein transport through the secretory pathway function exert their activities on the surfaces of membrane bilayers. It is now clear that the various lipid components of these bilayers play direct and versatile roles in modulating the activity of proteins that either themselves constitute core components of the membrane trafficking machinery, or represent proteins that regulate such core components.     

Phospholipid transfer activity is relevant to but not sufficient for the essential function of the yeast SEC14 gene product.

To investigate several key aspects of phosphatidylinositol transfer protein (PI-TP) function in eukaryotic cells, rat PI-TP was expressed in yeast strains carrying lesions in SEC14, the structural gene for yeast PI-TP (SEC14p), whose activity is essential for Golgi secretory function in vivo. Rat PI-TP expression effected a specific complementation of sec14ts growth and secretory defects. Complementation of sec14 mutations was not absolute as rat PI-TP expression failed to rescue sec14 null mutations. This partial complementation of sec14 lesions by rat PI-TP correlated with inability of the mammalian protein to stably associate with yeast Golgi membranes and was not a result of rat PI-TP stabilizing the endogenous sec14ts gene product. These collective data demonstrate that while the in vitro PI-TP activity of SEC14p clearly reflects some functional in vivo property of SEC14p, the PI-TP activity is not the sole essential activity of SEC14p. Those data further identify an efficient Golgi targeting capability as a likely essential feature of SEC14p function in vivo. Finally, the data suggest that stable association of SEC14p with yeast Golgi membranes is not a simple function of its lipid-binding properties, indicate that the amino-terminal 129 SEC14p residues are sufficient to direct a catalytically inactive form of rat PI-TP to the Golgi and provide the first evidence to indicate that a mammalian PI-TP can stimulate Golgi secretory function in vivo.     

The Saccharomyces cerevisiae SEC14 gene encodes a cytosolic factor that is required for transport of secretory proteins from the yeast Golgi complex

We have obtained and characterized a genomic clone of SEC14, a Saccharomyces cerevisiae gene whose product is required for export of yeast secretory proteins from the Golgi complex. Gene disruption experiments indicated that SEC14 is an essential gene for yeast vegetative growth. Nucleotide sequence analysis revealed the presence of an intron within the SEC14 structural gene, and predicted the synthesis of a hydrophilic polypeptide of 35 kD in molecular mass. In confirmation, immunoprecipitation experiments demonstrated SEC14p to be an unglycosylated polypeptide, with an apparent molecular mass of some 37 kD, that behaved predominantly as a cytosolic protein in subcellular fractionation experiments. These data were consistent with the notion that SEC14p is a cytosolic factor that promotes protein export from yeast Golgi. Additional radiolabeling experiments also revealed the presence of SEC14p-related polypeptides in extracts prepared from the yeasts Kluyveromyces lactis and Schizosaccharomyces pombe. Furthermore, the K. lactis SEC14p was able to functionally complement S. cerevisiae sec14ts defects. These data suggested a degree of conservation of SEC14p structure and function in these yeasts species.     

Activity of specific lipid-regulated ADP ribosylation factor-GTPase-activating proteins is required for Sec14p-dependent Golgi secretory function in yeast

Yeast phosphatidylinositol transfer protein (Sec14p) coordinates lipid metabolism with protein-trafficking events. This essential Sec14p requirement for Golgi function is bypassed by mutations in any one of seven genes that control phosphatidylcholine or phosphoinositide metabolism. In addition to these "bypass Sec14p" mutations, Sec14p-independent Golgi function requires phospholipase D activity. The identities of lipids that mediate Sec14p-dependent Golgi function, and the identity of the proteins that respond to Sec14p-mediated regulation of lipid metabolism, remain elusive. We now report genetic evidence to suggest that two ADP ribosylation factor-GTPase-activating proteins (ARFGAPs), Gcs1p and Age2p, may represent these lipid-responsive elements, and that Gcs1p/Age2p act downstream of Sec14p and phospholipase D in both Sec14p-dependent and Sec14p-independent pathways for yeast Golgi function. In support, biochemical data indicate that Gcs1p and Age2p ARFGAP activities are both modulated by lipids implicated in regulation of Sec14p pathway function. These results suggest ARFGAPs are stimulatory factors required for regulation of Golgi function by the Sec14p pathway, and that Sec14p-mediated regulation of lipid metabolism interfaces with the activity of proteins involved in control of the ARF cycle.     

Photolysis of caged sphingosine-1-phosphate induces barrier enhancement and intracellular activation of lung endothelial cell signaling pathways

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that mediates cellular functions by ligation via G protein-coupled S1P receptors. In addition to its extracellular action, S1P also has intracellular effects; however, the signaling pathways modulated by intracellular S1P remain poorly defined. We have previously demonstrated a novel pathway of intracellular S1P generation in human lung endothelial cells (ECs). In the present study, we examined the role of intracellular S1P generated by photolysis of caged S1P on EC barrier regulation and signal transduction. Intracellular S1P released from caged S1P caused mobilization of intracellular calcium, induced activation of MAPKs, redistributed cortactin, vascular endothelial cadherin, and β-catenin to cell periphery, and tightened endothelial barrier in human pulmonary artery ECs. Treatment of cells with pertussis toxin (PTx) had no effect on caged S1P-mediated effects on Ca(2+) mobilization, reorganization of cytoskeleton, cell adherens junction proteins, and barrier enhancement; however, extracellular S1P effects were significantly attenuated by PTx. Additionally, intracellular S1P also activated small GTPase Rac1 and its effector Ras GTPase-activating-like protein IQGAP1, suggesting involvement of these proteins in the S1P-mediated changes in cell-to-cell adhesion contacts. Downregulation of sphingosine kinase 1 (SphK1), but not SphK2, with siRNA or inhibition of SphK activity with an inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (CII) attenuated exogenously administrated S1P-induced EC permeability. Furthermore, S1P1 receptor inhibitor SB649164 abolished exogenous S1P-induced transendothelial resistance changes but had no effect on intracellular S1P generated by photolysis of caged S1P. These results provide evidence that intracellular S1P modulates signal transduction in lung ECs via signaling pathway(s) independent of S1P receptors.     

Conformational analysis of the telomerase RNA pseudoknot hairpin by Raman spectroscopy

We have measured the temperature-dependent Raman spectra of two 30-mer ribonucleotides that represent the wild-type (WT) and dyskeratosis congenita (DKC) mutant (MT) GC (107-108) --> AG structures of the pseudoknot hairpin region of human telomerase RNA. We have used these structures, previously characterized by UV-melting and NMR, as a model system for our Raman investigation. We observe that Raman hypochromism of vibrational bands, previously assigned to specific bases or conformational RNA markers, reflect temperature-dependent alterations in the pentaloop and stem structures of these two oligonucleotides. We also observe that the intense nu(s)(O-P-O) band at 812 cm(-1) indicates the presence of A-form backbone structure at relatively low temperatures in both the WT and MT RNA sequences. The mutation induces a decrease in the intensity of the uridine (rU) band at 1244 cm(-1) associated with C2'-endo/anti ribose conformation in the pentaloop. Two transition temperatures (T(m) ) were determined from the analysis of Raman difference intensity-temperature profiles of the 1256 cm(-1) band, which is associated with vibrations of cytidine (rC) residues, in particular, the C2'-endo/anti ribose conformation (T(m) 1 = 23.6 +/- 1.6 degrees C for WT and 19.7 +/- 2.8 degrees C for MT; T(m) 2 = 68.9 +/- 1.8 degrees C for WT and 70.9 +/- 1.1 degrees C for MT). From these results we can conclude that the DKC mutant 30-mer exhibits a lower stability in the pentaloop region and a slightly higher stability in the stem region than the WT 30-mer. This demonstrates that Raman bands, previously assigned to specific bases or conformational RNA markers, can be used to probe local structural features of the telomerase pseudoknot hairpin sequence.     

An innexin-dependent cell network establishes left-right neuronal asymmetry in C. elegans

Gap junctions are widespread in immature neuronal circuits, but their functional significance is poorly understood. We show here that a transient network formed by the innexin gap-junction protein NSY-5 coordinates left-right asymmetry in the developing nervous system of Caenorhabditis elegans. nsy-5 is required for the left and right AWC olfactory neurons to establish stochastic, asymmetric patterns of gene expression during embryogenesis. nsy-5-dependent gap junctions in the embryo transiently connect the AWC cell bodies with those of numerous other neurons. Both AWCs and several other classes of nsy-5-expressing neurons participate in signaling that coordinates left-right AWC asymmetry. The right AWC can respond to nsy-5 directly, but the left AWC requires nsy-5 function in multiple cells of the network. NSY-5 forms hemichannels and intercellular gap-junction channels in Xenopus oocytes, consistent with a combination of cell-intrinsic and network functions. These results provide insight into gap-junction activity in developing circuits.     

Lipid metabolism and regulation of membrane trafficking

The past 20 years have witnessed tremendous progress in our understanding of the molecular machinery that controls protein and membrane transport between organelles (Scheckman R, Orci L. Coat proteins and vesicle budding. Science 1996;271: 1526–1533 and Rothman JE. Mechanisms of intracellular protein transport. Nature 1994;372: 55–63.) The research efforts responsible for these impressive advances have largely focused on the identification and characterization of protein factors that participate in membrane trafficking events. The role of membranes and their lipid constituents has received considerably less attention. Indeed, until rather recently, popular models for mechanisms of membrane trafficking had relegated membrane lipids to the status of a passive platform, subject to deformation by the action of coat proteins whose polymerization and depolymerization govern vesicle budding and fusion reactions. The 1990s, and particularly its last half, has brought fundamental reappraisals of the interface of lipids and lipid metabolism in regulating intracellular membrane trafficking events. Some of the emerging themes are reviewed here.     

Translational control of lipogenic enzymes in the cell cycle of synchronous,growing yeast cells

Translational control during cell division determines when cells start a new cell cycle, how fast they complete it, the number of successive divisions, and how cells coordinate proliferation with available nutrients. The translational efficiencies of mRNAs in cells progressing synchronously through the mitotic cell cycle, while preserving the coupling of cell division with cell growth, remain uninvestigated. We now report comprehensive ribosome profiling of a yeast cell size series from the time of cell birth, to identify mRNAs under periodic translational control. The data reveal coordinate translational activation of mRNAs encoding lipogenic enzymes late in the cell cycle including Acc1p, the rate‐limiting enzyme acetyl‐CoA carboxylase. An upstream open reading frame (uORF) confers the translational control of ACC1 and adjusts Acc1p protein levels in different nutrients. The ACC1 uORF is relevant for cell division because its ablation delays cell cycle progression, reduces cell size, and suppresses the replicative longevity of cells lacking the Sch9p protein kinase regulator of ribosome biogenesis. These findings establish an unexpected relationship between lipogenesis and protein synthesis in mitotic cell divisions.     

Unsaturated fatty acid‐induced non‐canonical autophagy: unusual? or unappreciated?

The breakdown of cellular components via autophagy is crucial for cellular homeostasis. In this issue of The EMBO Journal, Niso‐Santano et al ( 2015 ) report the important observation that feeding cells with saturated or unsaturated fatty acids triggers mechanistically distinct autophagic responses. Feeding cells saturated fatty acid induced the canonical, BECN1/PI3K‐dependent autophagy pathway. Conversely, the unsaturated fatty acid oleate triggered autophagic responses that were independent of the BECN1/PI3K complex, but that required a functional Golgi system.