Identifying the mechanisms of eukaryotic genome evolution by comparative genomics is often complicated by the multiplicity of events that have taken place throughout the history of individual lineages, leaving only distorted and superimposed traces in the genome of each living organism. The hemiascomycete yeasts, with their compact genomes, similar lifestyle and distinct sexual and physiological properties, provide a unique opportunity to explore such mechanisms. We present here the complete, assembled genome sequences of four yeast species, selected to represent a broad evolutionary range within a single eukaryotic phylum, that after analysis proved to be molecularly as diverse as the entire phylum of chordates. A total of approximately 24,200 novel genes were identified, the translation products of which were classified together with Saccharomyces cerevisiae proteins into about 4,700 families, forming the basis for interspecific comparisons. Analysis of chromosome maps and genome redundancies reveal that the different yeast lineages have evolved through a marked interplay between several distinct molecular mechanisms, including tandem gene repeat formation, segmental duplication, a massive genome duplication and extensive gene loss.
Substantial efforts have recently been made to demonstrate the importance of lipids and lipid-modifying enzymes in various membrane trafficking processes, including calcium-regulated exocytosis of hormones and neurotransmitters. Among bioactive lipids, phosphatidic acid (PA) is an attractive candidate to promote membrane fusion through its ability to change membrane topology. To date, however, the biosynthetic pathway, the dynamic location, and actual function of PA in secretory cells remain unknown. Using a short interference RNA strategy on chromaffin and PC12 cells, we demonstrate here that phospholipase D1 is activated in secretagogue-stimulated cells and that it produces PA at the plasma membrane at the secretory granule docking sites. We show that phospholipase D1 activation and PA production represent key events in the exocytotic progression. Membrane capacitance measurements indicate that reduction of endogenous PA impairs the formation of fusion-competent granules. Finally, we show that the PLD1 short interference RNAmediated inhibition of exocytosis can be rescued by exogenous provision of a lipid that favors the transition of opposed bi-layer membranes to hemifused membranes having the outer leaflets fused. Our findings demonstrate that PA synthesis is required during exocytosis to facilitate a late event in the granule fusion pathway. We propose that the underlying mechanism is related to the ability of PA to alter membrane curvature and promote hemi-fusion. Phosphatidic acid (PA)2 is a pleiotropic bioactive lipid that has been proposed to activate selected enzymes (1), recruit proteins to membrane surfaces (2), and serve as a substrate for the formation of other signaling lipids (3). Most intriguingly, PA has also been shown to promote negative curvature in bi-layer membranes due to its small polar head-group in combination with two fatty-acyl side chains (4). The bulk of cellular PA is synthesized via two different acylation pathways, the glycerol 3-phosphate pathway and the dihydroxy acetone phosphate pathway, which are named according to their respective precursors. However, PA is also produced via hydrolysis of phosphatidylcholine by phospholipase D (PLD) (5) on a much faster time scale, and this latter source is thought to underlie the dynamic regulation of PA that allows it to function as a signaling lipid in agonist-stimulated cell biological responses such as secretion and changes in cellular morphology.In mammals, the classic PLD family is composed of a pair of membrane-associated proteins, PLD1 and PLD2. Both PLD isoforms require phosphatidylinositol 4,5-bisphosphate for their enzymatic activity. However, whereas PLD2 exhibits relatively high basal activity in isolation, full activation of PLD1 requires its stimulation by small GTPases of the ADP-ribosylation factor (ARF), Rho and Ral families, and protein kinase C (3, 6). PLD enzymes have been proposed to be involved in a number of cellular processes, including cell growth and survival, cell differentiation, and vesicular trafficking (3)....
Exocytosis of neurotransmitters and hormones occurs through the fusion of secretory vesicles with the plasma membrane. This highly regulated process involves key proteins, such as SNAREs, and specific lipids at the site of membrane fusion. Phospholipase D (PLD) has recently emerged as a promoter of membrane fusion in various exocytotic events potentially by providing fusogenic cone-shaped phosphatidic acid. We show here that PLD1 is regulated by ribosomal S6 kinase 2 (RSK2)-dependent phosphorylation. RSK2 is activated by a high K + -induced rise in cytosolic calcium. Expression of inactive RSK2 mutants or selective knockdown of endogenous RSK2 dramatically affects the different kinetic components of the exocytotic response in chromaffin cells. RSK2 physically interacts with and stimulates PLD activity through the phosphorylation of Thr-147 in the PLD1 amino-terminal phox homology domain. Expression of PLD1 phosphomimetic mutants fully restores secretion in cells depleted of RSK2, suggesting that RSK2 is a critical upstream signaling element in the activation of PLD1 to produce the lipids required for exocytosis. We propose that PLD-related defects in neuronal and endocrine activities could contribute to the effect observed after the loss-of-function mutations in Rsk2 that lead to Coffin–Lowry syndrome, an X-linked form of growth and mental retardation.
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