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In JoVE (1)
Other Publications (11)
Articles by Christopher Loewen in JoVE
JoVE General
A high-throughput method to globally study the organelle morphology in S. cerevisiae
Department of Cellular and Physiological Sciences, University of British Columbia - UBC
GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.
Other articles by Christopher Loewen on PubMed
Cholesterol Homeostasis: Not Until the SCAP Lady INSIGs
Three new studies have wide implications for cholesterol homeostasis, identifying a novel mechanism by which a sterol-sensing domain functions in the regulated activation of sterol regulatory element binding proteins.
A Conserved ER Targeting Motif in Three Families of Lipid Binding Proteins and in Opi1p Binds VAP
Intracellular lipid traffic is mediated both by membrane vesicles and by a number of non-vesicular pathways facilitated by cytoplasmic lipid binding proteins. For these proteins to act effectively they must be targeted accurately to specific membranes. Here we identify a novel short conserved determinant called the FFAT motif that is shared by several seemingly unrelated lipid binding proteins and is also found in Opi1p, a transcriptional regulator of phospholipid synthesis in yeast. FFAT motifs act as membrane- targeting determinants by their direct interaction with homologues of VAMP-associated protein (VAP), a conserved endoplasmic reticulum (ER) protein. In budding yeast, all four proteins with FFAT motifs interact with Scs2p, a homologue of VAP, to target the ER to some extent. The precise intracellular distribution of each of these proteins depends on the integration of the FFAT-Scs2p interaction with other targeting determinants, and the interaction is functionally significant. We conclude that binding to a VAP homologue is a common mechanism by which proteins with FFAT motifs, most of which are involved in lipid metabolism, target ER membranes.
The Role of Subunit Assembly in Peripherin-2 Targeting to Rod Photoreceptor Disk Membranes and Retinitis Pigmentosa
Peripherin-2 is a member of the tetraspanin family of membrane proteins that plays a critical role in photoreceptor outer segment disk morphogenesis. Mutations in peripherin-2 are responsible for various retinal degenerative diseases including autosomal dominant retinitis pigmentosa (ADRP). To identify determinants required for peripherin-2 targeting to disk membranes and elucidate mechanisms underlying ADRP, we have generated transgenic Xenopus tadpoles expressing wild-type and ADRP-linked peripherin-2 mutants as green fluorescent fusion proteins in rod photoreceptors. Wild-type peripherin-2 and P216L and C150S mutants, which assemble as tetramers, targeted to disk membranes as visualized by confocal and electron microscopy. In contrast the C214S and L185P mutants, which form homodimers, but not tetramers, were retained in the rod inner segment. Only the P216L disease mutant induced photoreceptor degeneration. These results indicate that tetramerization is required for peripherin-2 targeting and incorporation into disk membranes. Tetramerization-defective mutants cause ADRP through a deficiency in wild-type peripherin-2, whereas tetramerization-competent P216L peripherin-2 causes ADRP through a dominant negative effect, possibly arising from the introduction of a new oligosaccharide chain that destabilizes disks. Our results further indicate that a checkpoint between the photoreceptor inner and outer segments allows only correctly assembled peripherin-2 tetramers to be incorporated into nascent disk membranes.
Role of Subunit Assembly in Autosomal Dominant Retinitis Pigmentosa Linked to Mutations in Peripherin 2
Peripherin 2 is a photoreceptor-specific membrane protein implicated in outer segment disk morphogenesis and linked to various retinopathies including autosomal dominant retinitis pigmentosa (ADRP). Peripherin 2 and ROM1 assemble as a mixture of core noncovalent homomeric and heteromeric tetramers that further link together through disulfide bonds to form higher order oligomers. These complexes are critical for disk rim formation and outer segment structure through interaction with the cGMP-gated channel and other photoreceptor proteins. We have examined the role of subunit assembly in peripherin 2 targeting to disks, outer segment structure, and photoreceptor degeneration by examining molecular and cellular properties of peripherin 2 mutants in COS-1 cells and transgenic Xenopus laevis rod photoreceptors. Wild-type (WT) and the ADRP-linked P216L mutant were transported and incorporated into newly formed outer segment disks of transgenic X. laevis. The P216L mutant, however, induced progressive outer segment instability and photoreceptor degeneration possibly through the introduction of a new N-linked oligosaccharide chain. In contrast, the C214S and L185P disease-linked, tetramerization-defective mutants, were retained in the inner segment, but did not affect outer segment structure or induce photoreceptor degeneration. Together, these results indicate that peripherin 2 mutations can cause ADRP either through a deficiency in WT peripherin 2 (C214S, 1.185P) or by a dominant negative effect on disk stability (P216L).
A Highly Conserved Binding Site in Vesicle-associated Membrane Protein-associated Protein (VAP) for the FFAT Motif of Lipid-binding Proteins
A variety of lipid-binding proteins contain a recently described motif, designated FFAT (two phenylalanines in an acidic tract), which binds to vesicle-associated-membrane protein-associated protein (VAP). VAP is a conserved integral membrane protein of the endoplasmic reticulum that contains at its amino terminus a domain related to the major sperm protein of nematode worms. Here we have studied the FFAT-VAP interaction in Saccharomyces cerevisiae, where the VAP homologue Scs2 regulates phospholipid metabolism via an interaction with the FFAT motif of Opi1. By introducing mutations at random into Scs2, we found that mutations that abrogated binding to FFAT were clustered in the most highly conserved region. Using site-directed mutagenesis, we identified several critical residues, including two lysines widely separated in the primary sequence. By examining all other conserved basic residues, we identified a third residue that was moderately important for binding FFAT. Modeling VAP on the known structure of major sperm protein showed that the critical residues form a patch on a positively charged face of the protein. In vivo functional studies of SCS22, a second SCS2-like gene in S. cerevisiae, showed that SCS2 was the dominant gene in the regulation of Opi1, with a minor contribution from SCS22. We then established that reduction in the affinity of Scs2 mutants for FFAT correlated well with loss of function, indicating the importance of these residues for binding FFAT motifs. Finally, we found that human VAP-A could substitute for Scs2 but that it functioned poorly, suggesting that other factors modulate the binding of Scs2 to proteins with FFAT motifs.
Inheritance of Cortical ER in Yeast is Required for Normal Septin Organization
How cells monitor the distribution of organelles is largely unknown. In budding yeast, the largest subdomain of the endoplasmic reticulum (ER) is a network of cortical ER (cER) that adheres to the plasma membrane. Delivery of cER from mother cells to buds, which is termed cER inheritance, occurs as an orderly process early in budding. We find that cER inheritance is defective in cells lacking Scs2, a yeast homologue of the integral ER membrane protein VAP (vesicle-associated membrane protein-associated protein) conserved in all eukaryotes. Scs2 and human VAP both target yeast bud tips, suggesting a conserved action of VAP in attaching ER to sites of polarized growth. In addition, the loss of either Scs2 or Ice2 (another protein involved in cER inheritance) perturbs septin assembly at the bud neck. This perturbation leads to a delay in the transition through G2, activating the Saccharomyces wee1 kinase (Swe1) and the morphogenesis checkpoint. Thus, we identify a mechanism involved in sensing the distribution of ER.
Critical Determinants for Chromatin Binding by Saccharomyces Cerevisiae Yng1 Exist Outside of the Plant Homeodomain Finger
The temporal and spatial regulation of histone post-translational modifications is essential for proper chromatin structure and function. The Saccharomyces cerevisiae NuA3 histone acetyltransferase complex modifies the amino-terminal tail of histone H3, but how NuA3 is targeted to specific regions of the genome is not fully understood. Yng1, a subunit of NuA3 and a member of the Inhibitor of Growth (ING) protein family, is required for the interaction of NuA3 with chromatin. This protein contains a C-terminal plant homeodomain (PHD) finger that specifically interacts with lysine 4-trimethylated histone H3 (H3K4me3) in vitro. This initially suggested that NuA3 is targeted to regions bearing the H3K4me3 mark; however, deletion of the Yng1 PHD finger does not disrupt the interaction of NuA3 with chromatin or result in a phenotype consistent with loss of NuA3 function in vivo. In this study, we uncovered the molecular basis for the discrepancies in these data. We present both genetic and biochemical evidence that full-length Yng1 has two independent histone-binding motifs: an amino-terminal motif that binds unmodified H3 tails and a carboxyl-terminal PHD finger that specifically recognizes H3K4me3. Although these motifs can bind histones independently, together they increase the apparent association of Yng1 for the H3 tail.
Phosphatidic Acid is a PH Biosensor That Links Membrane Biogenesis to Metabolism
Recognition of lipids by proteins is important for their targeting and activation in many signaling pathways, but the mechanisms that regulate such interactions are largely unknown. Here, we found that binding of proteins to the ubiquitous signaling lipid phosphatidic acid (PA) depended on intracellular pH and the protonation state of its phosphate headgroup. In yeast, a rapid decrease in intracellular pH in response to glucose starvation regulated binding of PA to a transcription factor, Opi1, that coordinately repressed phospholipid metabolic genes. This enabled coupling of membrane biogenesis to nutrient availability.
High Density Array Screening to Identify the Genetic Requirements for Transition Metal Tolerance in Saccharomyces Cerevisiae
Biological systems have developed with a strong dependence on transition metals for accomplishing a number of biochemical reactions. Iron, copper, manganese and zinc are essential for virtually all forms of life with their unique chemistries contributing to a variety of physiological processes including oxygen transport, generation of cellular energy and protein structure and function. Properties of these metals (and to a lesser extent nickel and cobalt) that make them so essential to life also make them extremely cytotoxic in many cases through the formation of damaging oxygen radicals via Fenton chemistry. While life has evolved to exploit the chemistries of transition metals to drive physiological reactions, systems have concomitantly evolved to protect against the damaging effects of these same metals. Saccharomyces cerevisiae is a valuable tool for studying metal homeostasis with many of the genes identified thus far having homologs in higher eukaryotes including humans. Using high density arrays, we have screened a haploid S. cerevisiae deletion set containing 4786 non-essential gene deletions for strains sensitive to each of Fe, Cu, Mn, Ni, Zn and Co and then integrated the six screens using cluster analysis to identify pathways that are unique to individual metals and others with function shared between metals. Genes with no previous implication in metal homeostasis were found to contribute to sensitivity to each metal. Significant overlap was observed between the strains that were sensitive to Mn, Ni, Zn and Co with many of these strains lacking genes for the high affinity Fe transport pathway and genes involved in vacuolar transport and acidification. The results from six genome-wide metal tolerance screens show that there is some commonality between the cellular defenses against the toxicity of Mn, Ni, Zn and Co with Fe and Cu requiring different systems. Additionally, potential new factors been identified that function in tolerance to each of the six metals.
Putting the PH into Phosphatidic Acid Signaling
The lipid phosphatidic acid (PA) has important roles in cell signaling and metabolic regulation in all organisms. New evidence indicates that PA also has an unprecedented role as a pH biosensor, coupling changes in pH to intracellular signaling pathways. pH sensing is a property of the phosphomonoester headgroup of PA. A number of other potent signaling lipids also contain headgroups with phosphomonoesters, implying that pH sensing by lipids may be widespread in biology.
Lipids As Conductors in the Orchestra of Life
The lipid phosphatidic acid is an important metabolic intermediate in the biosynthesis of lipids in all eukaryotic cells, but it is even more than that. Phosphatidic acid is emerging as a lipid that is both composer and conductor, where in addition to its role as biosynthetic precursor (composer) it is also a potent signaling molecule (conductor) that integrates membrane biogenesis with nutrient sensing and cell growth. This article discusses recent advances in yeast that give praise for phosphatidic acid as one of life's conductors.
