Role of Sec16p in the formation of COPII-coated ER to Goldi transport vesicles in Saccharomyces cerevisiae

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 1998.Includes bibliographical references.by Peter J. Espendhsade.Ph.D

SREBP Controls Oxygen-Dependent Mobilization of Retrotransposons in Fission Yeast

Retrotransposons are mobile genetic elements that proliferate through an RNA intermediate. Transposons do not encode transcription factors and thus rely on host factors for mRNA expression and survival. Despite information regarding conditions under which elements are upregulated, much remains to be learned about the regulatory mechanisms or factors controlling retrotransposon expression. Here, we report that low oxygen activates the fission yeast Tf2 family of retrotransposons. Sre1, the yeast ortholog of the mammalian membrane-bound transcription factor sterol regulatory element binding protein (SREBP), directly induces the expression and mobilization of Tf2 retrotransposons under low oxygen. Sre1 binds to DNA sequences in the Tf2 long terminal repeat that functions as an oxygen-dependent promoter. We find that Tf2 solo long terminal repeats throughout the genome direct oxygen-dependent expression of adjacent coding and noncoding sequences, providing a potential mechanism for the generation of oxygen-dependent gene expression

Sterol regulatory element-binding protein (Sre1) promotes the synthesis of carotenoids and sterols in xanthophyllomyces dendrorhous

Xanthophyllomyces dendrorhous is a basidiomycete yeast that synthesizes carotenoids, mainly astaxanthin, which are of great commercial interest. Currently, there are many unknown aspects related to regulatory mechanisms on the synthesis of carotenoids in this yeast. Our recent studies showed that changes in sterol levels and composition resulted in upregulation of genes in the mevalonate pathway required for the synthesis of carotenoid precursors, leading to increased production of these pigments. Sterol Regulatory Element-Binding Proteins (SREBP), called Sre1 in yeast, are conserved transcriptional regulators of sterol homeostasis and other cellular processes. Given the results linking sterols and carotenoids, we investigated the role of SREBP in sterol and carotenoid synthesis in X. dendrorhous. In this study, we present the identification and functional characterization of the X. dendrorhous SRE1 gene, which encodes the transcription factor Sre1. The deduced protein has the characteristic features of SREBP/Sre1 and binds to consensus DNA sequences in vitro. RNA-seq analysis and chromatin-immunoprecipitation experiments showed that genes of the mevalonate pathway and ergosterol biosynthesis are directly regulated by Sre1. The sre1- mutation reduced sterol and carotenoid production in X. dendrorhous, and expression of the Sre1 N-terminal domain (Sre1N) increased carotenoid production more than twofold compared to wild-type. Overall, our results indicate that in X. dendrorhous transcriptional regulation of genes in the mevalonate pathway control production of the isoprenoid derivatives, carotenoids and sterol. Our results provide new insights into the conserved regulatory functions of SREBP/Sre1 and identify pointing to the SREBP pathway as a potential target to enhance carotenoid production in X. dendrorhous. domain (Sre1N) increased carotenoid production more than twofold compared to wild-type. Overall, our results indicate that in X. dendrorhous transcriptional regulation of genes in the mevalonate pathway control production of the isoprenoid derivatives, carotenoids and sterol. Our results provide new insights into the conserved regulatory functions of SREBP/Sre1 and identify pointing to the SREBP pathway as a potential target to enhance carotenoid production in X. dendrorhous.FONDECYT 1160202 and by graduate scholarships CONICYT 21130708 to MSG and CONICYT 2117061

Sec16 Defines Endoplasmic Reticulum Exit Sites and is Required for Secretory Cargo Export in Mammalian Cells

The selective export of proteins and lipids from the endoplasmic reticulum (ER) is mediated by the coat protein complex II (COPII) that assembles onto the ER membrane. In higher eukaryotes, COPII proteins assemble at discrete sites on the membrane known as ER exit sites (ERES). Here, we identify Sec16 as the protein that defines ERES in mammalian cells. Sec16 localizes to ERES independent of Sec23/24 and Sec13/31. Overexpression, and to a lesser extent, small interfering RNA depletion of Sec16, both inhibit ER-to-Golgi transport suggesting that Sec16 is required in stoichiometric amounts. Sar1 activity is required to maintain the localization of Sec16 at discrete locations on the ER membrane, probably through preventing its dissociation. Our data suggest that Sar1-GTP-dependent assembly of Sec16 on the ER membrane forms an organized scaffold defining an ERES

Sterol Regulatory Element-Binding Protein (Sre1) Promotes the Synthesis of Carotenoids and Sterols in Xanthophyllomyces dendrorhous

Xanthophyllomyces dendrorhous is a basidiomycete yeast that synthesizes carotenoids, mainly astaxanthin, which are of great commercial interest. Currently, there are many unknown aspects related to regulatory mechanisms on the synthesis of carotenoids in this yeast. Our recent studies showed that changes in sterol levels and composition resulted in upregulation of genes in the mevalonate pathway required for the synthesis of carotenoid precursors, leading to increased production of these pigments. Sterol Regulatory Element-Binding Proteins (SREBP), called Sre1 in yeast, are conserved transcriptional regulators of sterol homeostasis and other cellular processes. Given the results linking sterols and carotenoids, we investigated the role of SREBP in sterol and carotenoid synthesis in X.dendrorhous. In this study, we present the identification and functional characterization of the X.dendrorhousSRE1 gene, which encodes the transcription factor Sre1. The deduced protein has the characteristic features of SREBP/Sre1 and binds to consensus DNA sequences in vitro. RNA-seq analysis and chromatin-immunoprecipitation experiments showed that genes of the mevalonate pathway and ergosterol biosynthesis are directly regulated by Sre1. The sre1- mutation reduced sterol and carotenoid production in X. dendrorhous, and expression of the Sre1 N-terminal domain (Sre1N) increased carotenoid production more than twofold compared to wild-type. Overall, our results indicate that in X. dendrorhous transcriptional regulation of genes in the mevalonate pathway control production of the isoprenoid derivatives, carotenoids and sterol. Our results provide new insights into the conserved regulatory functions of SREBP/Sre1 and identify pointing to the SREBP pathway as a potential target to enhance carotenoid production in X. dendrorhous

Sterols regulate ER-to-Golgi transport of SREBP cleavage activating protein (SCAP)

Presents Cholesterol homeostasis in mammalian cells is regulated at the level of transcription through feedback inhibition. When cells are depleted of sterols, transcription of genes required for synthesis and uptake of cholesterol, such as HMG-CoA reductase (HMGR) and LDL receptor, is upregulated by a family of ER membrane-bound transcription factors called sterol regulatory element binding proteins, SREBP. The N terminus of SREBP is a basic helix-loop-helix leucine zipper transcription factor and the C terminus binds tightly to SREBP cleavage activating protein (SCAP). In sterol-replete cells, the SREBP-SCAP complex remains in the ER, compartmentally separated from the Golgi-localized Site-1 and Site-2 proteases. ER retention of SREBP-SCAP is mediated by the sterol-dependent binding of SREBP-SCAP to the ER resident protein Insig. In the first video, sterol-replete Chinese Hamster Ovary cells stably expressing GFP-SCAP were imaged in the presence of excess sterol that inhibits ER-to-Golgi transport of SCAP. In sterol-depleted cells, SCAP does not bind Insig, allowing ER exit of SREBP-SCAP via COPII vesicles. Transport to the Golgi results in SREBP cleavage by Site-1 and Site-2 proteases and release of the N-terminal transcription factor domain from the membrane. Activated SREBP enters the nucleus and upregulates genes required for cholesterol supply. Following SREBP cleavage, SCAP is thought to recycle from the Golgi to the ER in order to participate in additional rounds of SREBP proteolysis. In the second video, cells were acutely depleted of cholesterol at t=0 to induce SCAP transport to the Golgi. GFP-SCAP first appears in vesicular structures and then clearly accumulates in the Golgi by t=15 minComponente Curricular::Educação Superior::Ciências Biológicas::Biologia Gera

Sterols regulate ER-to-Golgi transport of SREBP cleavage activating protein (SCAP)

Presents Cholesterol homeostasis in mammalian cells is regulated at the level of transcription through feedback inhibition. When cells are depleted of sterols, transcription of genes required for synthesis and uptake of cholesterol, such as HMG-CoA reductase (HMGR) and LDL receptor, is upregulated by a family of ER membrane-bound transcription factors called sterol regulatory element binding proteins, SREBP. The N terminus of SREBP is a basic helix-loop-helix leucine zipper transcription factor and the C terminus binds tightly to SREBP cleavage activating protein (SCAP). In sterol-replete cells, the SREBP-SCAP complex remains in the ER, compartmentally separated from the Golgi-localized Site-1 and Site-2 proteases. ER retention of SREBP-SCAP is mediated by the sterol-dependent binding of SREBP-SCAP to the ER resident protein Insig. In the first video, sterol-replete Chinese Hamster Ovary cells stably expressing GFP-SCAP were imaged in the presence of excess sterol that inhibits ER-to-Golgi transport of SCAP. In sterol-depleted cells, SCAP does not bind Insig, allowing ER exit of SREBP-SCAP via COPII vesicles. Transport to the Golgi results in SREBP cleavage by Site-1 and Site-2 proteases and release of the N-terminal transcription factor domain from the membrane. Activated SREBP enters the nucleus and upregulates genes required for cholesterol supply. Following SREBP cleavage, SCAP is thought to recycle from the Golgi to the ER in order to participate in additional rounds of SREBP proteolysis. In the second video, cells were acutely depleted of cholesterol at t=0 to induce SCAP transport to the Golgi. GFP-SCAP first appears in vesicular structures and then clearly accumulates in the Golgi by t=15 minComponente Curricular::Educação Superior::Ciências Biológicas::Biologia Gera