Optimization of Tabersonine Methoxylation to Increase Vindoline Precursor Synthesis in Yeast Cell Factories.

Plant specialized metabolites are widely used in the pharmaceutical industry, including the monoterpene indole alkaloids (MIAs) vinblastine and vincristine, which both display anticancer activity. Both compounds can be obtained through the chemical condensation of their precursors vindoline and catharanthine extracted from leaves of the Madagascar periwinkle. However, the extensive use of these molecules in chemotherapy increases precursor demand and results in recurrent shortages, explaining why the development of alternative production approaches, such microbial cell factories, is mandatory. In this context, the precursor-directed biosynthesis of vindoline from tabersonine in yeast-expressing heterologous biosynthetic genes is of particular interest but has not reached high production scales to date. To circumvent production bottlenecks, the metabolic flux was channeled towards the MIA of interest by modulating the copy number of the first two genes of the vindoline biosynthetic pathway, namely tabersonine 16-hydroxylase and tabersonine-16-O-methyltransferase. Increasing gene copies resulted in an optimized methoxylation of tabersonine and overcame the competition for tabersonine access with the third enzyme of the pathway, tabersonine 3-oxygenase, which exhibits a high substrate promiscuity. Through this approach, we successfully created a yeast strain that produces the fourth biosynthetic intermediate of vindoline without accumulation of other intermediates or undesired side-products. This optimization will probably pave the way towards the future development of yeast cell factories to produce vindoline at an industrial scale.

Enhanced bioproduction of anticancer precursor vindoline by yeast cell factories.

The pharmaceutical industry faces a growing demand and recurrent shortages in many anticancer plant drugs given their extensive use in human chemotherapy. Efficient alternative strategies of supply of these natural products such as bioproduction by microorganisms are needed to ensure stable and massive manufacturing. Here, we developed and optimized yeast cell factories efficiently converting tabersonine to vindoline, a precursor of the major anticancer alkaloids vinblastine and vincristine. First, fine-tuning of heterologous gene copies restrained side metabolites synthesis towards vindoline production. Tabersonine to vindoline bioconversion was further enhanced through a rational medium optimization (pH, composition) and a sequential feeding strategy. Finally, a vindoline titre of 266 mg l-1 (88% yield) was reached in an optimized fed-batch bioreactor. This precursor-directed synthesis of vindoline thus paves the way towards future industrial bioproduction through the valorization of abundant tabersonine resources.

Cex1 is a component of the COPI intracellular trafficking machinery.

COPI (coatomer complex I) coated vesicles are involved in Golgi-to-ER and intra-Golgi trafficking pathways, and mediate retrieval of ER resident proteins. Functions and components of the COPI-mediated trafficking pathways, beyond the canonical set of Sec/Arf proteins, are constantly increasing in number and complexity. In mammalian cells, GORAB, SCYL1 and SCYL3 proteins regulate Golgi morphology and protein glycosylation in concert with the COPI machinery. Here, we show that Cex1, homologous to the mammalian SCYL proteins, is a component of the yeast COPI machinery, by interacting with Sec27, Sec28 and Sec33 (Ret1/Cop1) proteins of the COPI coat. Cex1 was initially reported to mediate channeling of aminoacylated tRNA outside of the nucleus. Our data show that Cex1 localizes at membrane compartments, on structures positive for the Sec33 α-COP subunit. Moreover, the Wbp1 protein required for N-glycosylation and interacting via its di-lysine motif with the Sec27 ß'-COP subunit is mis-targeted in cex1Δ deletion mutant cells. Our data point to the possibility of developing Cex1 yeast-based models to study neurodegenerative disorders linked to pathogenic mutations of its human homologue SCYL1.

Exploiting Spermidine N-Hydroxycinnamoyltransferase Diversity and Substrate Promiscuity to Produce Various Trihydroxycinnamoyl Spermidines and Analogues in Engineered Yeast.

Trihydroxycinnamoyl spermidines (THCSpd) are plant specialized metabolites with promising pharmacological activities as antifungals, antibacterial, antiviral, and antidepressant drugs. However, their characterization and potential pharmaceutical exploitation are greatly impaired by the sourcing of these compounds, restricted to the pollen of core Eudicot plant species. In this work, we developed a precursor-directed biosynthesis of THCSpd in yeast using a dual enzymatic system based on 4-coumarate-CoA ligases (4CL) and spermidine N-hydroxycinnamoyltransferases (SHT). The system relies on the yeast endogenous spermidine pool and only requires hydroxycinnamic acids as exogenous precursors. By exploring 4CL isoforms and SHT diversity among plants, we have driven the production of 8 natural THCSpd, using single or mixed hydroxycinnamic acid precursors. Substrate promiscuities of 4CL and SHT were genuinely exploited to produce 8 new-to-nature THCSpd from exotic hydroxycinnamic and dihydrohydroxycinnamic acids, together with 3 new-to-nature THCSpd containing halogenated hydroxycinnamoyl moieties. In this work, we established a versatile and modular biotechnological production platform allowing the tailor-made THCSpd synthesis, constituting pioneer metabolic engineering for access to these valuable natural products.

Assigning mitochondrial localization of dual localized proteins using a yeast Bi-Genomic Mitochondrial-Split-GFP.

A single nuclear gene can be translated into a dual localized protein that distributes between the cytosol and mitochondria. Accumulating evidences show that mitoproteomes contain lots of these dual localized proteins termed echoforms. Unraveling the existence of mitochondrial echoforms using current GFP (Green Fluorescent Protein) fusion microscopy approaches is extremely difficult because the GFP signal of the cytosolic echoform will almost inevitably mask that of the mitochondrial echoform. We therefore engineered a yeast strain expressing a new type of Split-GFP that we termed Bi-Genomic Mitochondrial-Split-GFP (BiG Mito-Split-GFP). Because one moiety of the GFP is translated from the mitochondrial machinery while the other is fused to the nuclear-encoded protein of interest translated in the cytosol, the self-reassembly of this Bi-Genomic-encoded Split-GFP is confined to mitochondria. We could authenticate the mitochondrial importability of any protein or echoform from yeast, but also from other organisms such as the human Argonaute 2 mitochondrial echoform.

Improved gene co-expression network quality through expression dataset down-sampling and network aggregation.

Large-scale gene co-expression networks are an effective methodology to analyze sets of co-expressed genes and discover new gene functions or associations. Distances between genes are estimated according to their expression profiles and are visualized in networks that may be further partitioned to reveal communities of co-expressed genes. Creating expression profiles is now eased by the large amounts of publicly available expression data (microarrays and RNA-seq). Although many distance calculation methods have been intensively compared and reviewed in the past, it is unclear how to proceed when many samples reflecting a wide range of different conditions are available. Should as many samples as possible be integrated into network construction or be partitioned into smaller sets of more related samples? Previous studies have indicated a saturation in network performances to capture known associations once a certain number of samples is included in distance calculations. Here, we examined the influence of sample size on co-expression network construction using microarray and RNA-seq expression data from three plant species. We tested different down-sampling methods and compared network performances in recovering known gene associations to networks obtained from full datasets. We further examined how aggregating networks may help increase this performance by testing six aggregation methods.

Genome-wide identification and biochemical characterization of the UGT88F subfamily in Malus x domestica Borkh.

The UDP-glycosyltransferase UGT88F subfamily has been described first in Malus x domestica with the characterization of UGT88F1. Up to now UGT88F1 was one of the most active UGT glycosylating dihydrochalcones in vitro. The involvement of UGT88F1 in phloridzin (phloretin 2'-O-glucoside) synthesis, the main apple tree dihydrochalcone, was further confirmed in planta. Since the characterization of UGT88F1, this new UGT subfamily has been poorly studied probably because it seemed restricted to Maloideae. In the present study, we investigate the apple tree genome to identify and biochemically characterize the whole UGT88F subfamily. The apple tree genome contains five full-length UGT88F genes out of which three newly identified members (UGT88F6, UGT88F7 and UGT88F8) and a pseudogene. These genes are organized into two genomic clusters resulting from the recent global genomic duplication event in the apple tree. We show that recombinant UGT88F8 protein specifically glycosylates phloretin in the 2'OH position to synthetize phloridzin in vitro and was therefore named UDP-glucose: phloretin 2'-O-glycosyltransferase. The Km values of UGT88F8 are 7.72 µM and 10.84 µM for phloretin and UDP-glucose respectively and are in the same range as UGT88F1 catalytic parameters thus constituting two isoforms. Co-expression patterns of both UGT88F1 and UGT88F8 argue for a redundant function in phloridzin biosynthesis in planta. Contrastingly, recombinant UGT88F6 protein is able to glycosylate in vitro a wide range of flavonoids including flavonols, flavones, flavanones, chalcones and dihydrochalcones, although flavonols are the preferred substrates, e.g. Km value for kaempferol is 2.1 µM. Depending on the flavonoid, glycosylation occurs at least on the 3-OH and 7-OH positions. Therefore UGT88F6 corresponds to an UDP-glucose: flavonoid 3/7-O-glycosyltransferase. Finally, a molecular modeling study highlights a very high substitution rate of residues in the acceptor binding pocket between UGT88F8 and UGT88F6 which is responsible for the enzymes divergence in substrate and regiospecificity, despite an overall high protein homology.

Ranking genome-wide correlation measurements improves microarray and RNA-seq based global and targeted co-expression networks.

Co-expression networks are essential tools to infer biological associations between gene products and predict gene annotation. Global networks can be analyzed at the transcriptome-wide scale or after querying them with a set of guide genes to capture the transcriptional landscape of a given pathway in a process named Pathway Level Coexpression (PLC). A critical step in network construction remains the definition of gene co-expression. In the present work, we compared how Pearson Correlation Coefficient (PCC), Spearman Correlation Coefficient (SCC), their respective ranked values (Highest Reciprocal Rank (HRR)), Mutual Information (MI) and Partial Correlations (PC) performed on global networks and PLCs. This evaluation was conducted on the model plant Arabidopsis thaliana using microarray and differently pre-processed RNA-seq datasets. We particularly evaluated how dataset × distance measurement combinations performed in 5 PLCs corresponding to 4 well described plant metabolic pathways (phenylpropanoid, carbohydrate, fatty acid and terpene metabolisms) and the cytokinin signaling pathway. Our present work highlights how PCC ranked with HRR is better suited for global network construction and PLC with microarray and RNA-seq data than other distance methods, especially to cluster genes in partitions similar to biological subpathways.

Two Tabersonine 6,7-Epoxidases Initiate Lochnericine-Derived Alkaloid Biosynthesis in Catharanthus roseus.

Lochnericine is a major monoterpene indole alkaloid (MIA) in the roots of Madagascar periwinkle (Catharanthus roseus). Lochnericine is derived from the stereoselective C6,C7-epoxidation of tabersonine and can be metabolized further to generate other complex MIAs. While the enzymes responsible for its downstream modifications have been characterized, those involved in lochnericine biosynthesis remain unknown. By combining gene correlation studies, functional assays, and transient gene inactivation, we identified two highly conserved P450s that efficiently catalyze the epoxidation of tabersonine: tabersonine 6,7-epoxidase isoforms 1 and 2 (TEX1 and TEX2). Both proteins are quite divergent from the previously characterized tabersonine 2,3-epoxidase and are more closely related to tabersonine 16-hydroxylase, involved in vindoline biosynthesis in leaves. Biochemical characterization of TEX1/2 revealed their strict substrate specificity for tabersonine and their inability to epoxidize 19-hydroxytabersonine, indicating that they catalyze the first step in the pathway leading to hörhammericine production. TEX1 and TEX2 displayed complementary expression profiles, with TEX1 expressed mainly in roots and TEX2 in aerial organs. Our results suggest that TEX1 and TEX2 originated from a gene duplication event and later acquired divergent, organ-specific regulatory elements for lochnericine biosynthesis throughout the plant, as supported by the presence of lochnericine in flowers. Finally, through the sequential expression of TEX1 and up to four other MIA biosynthetic genes in yeast, we reconstituted the 19-acetylhörhammericine biosynthetic pathway and produced tailor-made MIAs by mixing enzymatic modules that are naturally spatially separated in the plant. These results lay the groundwork for the metabolic engineering of tabersonine/lochnericine derivatives of pharmaceutical interest.

Phosphoinositides, Major Actors in Membrane Trafficking and Lipid Signaling Pathways.

Phosphoinositides are lipids involved in the vesicular transport of proteins and lipids between the different compartments of eukaryotic cells. They act by recruiting and/or activating effector proteins and thus are involved in regulating various cellular functions, such as vesicular budding, membrane fusion and cytoskeleton dynamics. Although detected in small concentrations in membranes, their role is essential to cell function, since imbalance in their concentrations is a hallmark of many cancers. Their synthesis involves phosphorylating/dephosphorylating positions D3, D4 and/or D5 of their inositol ring by specific lipid kinases and phosphatases. This process is tightly regulated and specific to the different intracellular membranes. Most enzymes involved in phosphoinositide synthesis are conserved between yeast and human, and their loss of function leads to severe diseases (cancer, myopathy, neuropathy and ciliopathy).

Nonconventional localizations of cytosolic aminoacyl-tRNA synthetases in yeast and human cells.

By definition, cytosolic aminoacyl-tRNA synthetases (aaRSs) should be restricted to the cytosol of eukaryotic cells where they supply translating ribosomes with their aminoacyl-tRNA substrates. However, it has been shown that other translationally-active compartments like mitochondria and plastids can simultaneously contain the cytosolic aaRS and its corresponding organellar ortholog suggesting that both forms do not share the same organellar function. In addition, a fair number of cytosolic aaRSs have also been found in the nucleus of cells from several species. Hence, these supposedly cytosolic-restricted enzymes have instead the potential to be multi-localized. As expected, in all examples that were studied so far, when the cytosolic aaRS is imported inside an organelle that already contains its bona fide corresponding organellar-restricted aaRSs, the cytosolic form was proven to exert a nonconventional and essential function. Some of these essential functions include regulating homeostasis and protecting against various stresses. It thus becomes critical to assess meticulously the subcellular localization of each of these cytosolic aaRSs to unravel their additional roles. With this objective in mind, we provide here a review on what is currently known about cytosolic aaRSs multi-compartmentalization and we describe all commonly used protocols and procedures for identifying the compartments in which cytosolic aaRSs relocalize in yeast and human cells.

A mutation in VPS15 (PIK3R4) causes a ciliopathy and affects IFT20 release from the cis-Golgi.

Ciliopathies are a group of diseases that affect kidney and retina among other organs. Here, we identify a missense mutation in PIK3R4 (phosphoinositide 3-kinase regulatory subunit 4, named VPS15) in a family with a ciliopathy phenotype. Besides being required for trafficking and autophagy, we show that VPS15 regulates primary cilium length in human fibroblasts, as well as ciliary processes in zebrafish. Furthermore, we demonstrate its interaction with the golgin GM130 and its localization to the Golgi. The VPS15-R998Q patient mutation impairs Golgi trafficking functions in humanized yeast cells. Moreover, in VPS15-R998Q patient fibroblasts, the intraflagellar transport protein IFT20 is not localized to vesicles trafficking to the cilium but is restricted to the Golgi. Our findings suggest that at the Golgi, VPS15 and GM130 form a protein complex devoid of VPS34 to ensure the IFT20-dependent sorting and transport of membrane proteins from the cis-Golgi to the primary cilium.

[Phosphoinositides: lipidic essential actors in the intracellular traffic]. / Les phosphoinositides, des lipides acteurs essentiels du trafic intracellulaire.

Phosphoinositides (PPIn) are lipids involved in the vesicular transport of proteins between the different intracellular compartments. They act by recruiting and/or activating effector proteins and are thus involved in crucial cellular functions including vesicle budding, fusion and dynamics of membranes and regulation of the cytoskeleton. Although they are present in low concentrations in membranes, their activity is essential for cell survival and needs to be tightly controlled. Therefore, phosphatases and kinases specific of the various cellular membranes can phosphorylate/dephosphorylate their inositol ring on the positions D3, D4 and/or D5. The differential phosphorylation determines the intracellular localisation and the activity of the PPIn. Indeed, non-phosphorylated phosphatidylinositol (PtdIns) is the basic component of the PPIn and can be found in all eukaryotic cells at the cytoplasmic face of the ER, the Golgi, mitochondria and microsomes. It can get phosphorylated on position D4 to obtain PtdIns4P, a PPIn enriched in the Golgi compartment and involved in the maintenance of this organelle as well as anterograde and retrograde transport to and from the Golgi. PtdIns phosphorylation on position D3 results in PtdIns3P that is required for endosomal transport and multivesicular body (MVB) formation and sorting. These monophosphorylated PtdIns can be further phosphorylated to produce bisphophorylated PtdIns. Thus, PtdIns(4,5)P2, mainly produced by PtdIns4P phosphorylation, is enriched in the plasma membrane and involved in the regulation of actin cytoskeleton and endocytosis. PtdIns(3,5)P2, mainly produced by PtdIns3P phosphorylation, is enriched in late endosomes, MVBs and the lysosome/vacuole and plays a role in endosome to vacuole transport. PtdIns(3,4)P2 is absent in yeast, cells and mainly produced by PtdIns4P phosphorylation in human cells; PtdIns(3,4)P2 is localised in the plasma membrane and plays an important role as a second messenger by recruiting specific protein kinases (Akt and PDK1). Finally the triple phosphorylated PPIn, PtdIns(3,4,5)P3 also absent in yeast, is produced by the phosphorylation of PtdIns(3,4)P2 and localized at the plasma membrane of human cells where it binds proteins via their PH domain. Interaction partners include members of the Arf (ADP-ribosylation factors) family, PDK1 (Phosphoinositide Dependent Kinase 1) and Akt. Therefore this last PPIn is essential for the control of cell proliferation and its deregulation leads to the development of numerous cancers. In conclusion, the regulation of PPIn phosphorylation/dephosphorylation is complex and needs to be very precisely regulated. Indeed phosphatases and kinases allow the maintenance of the equilibrium between the different forms. PPIn play a crucial role in numerous cellular functions and a loss in their synthesis or regulation results in severe genetic diseases.

Membrane trafficking in the yeast Saccharomyces cerevisiae model.

The yeast Saccharomyces cerevisiae is one of the best characterized eukaryotic models. The secretory pathway was the first trafficking pathway clearly understood mainly thanks to the work done in the laboratory of Randy Schekman in the 1980s. They have isolated yeast sec mutants unable to secrete an extracellular enzyme and these SEC genes were identified as encoding key effectors of the secretory machinery. For this work, the 2013 Nobel Prize in Physiology and Medicine has been awarded to Randy Schekman; the prize is shared with James Rothman and Thomas Südhof. Here, we present the different trafficking pathways of yeast S. cerevisiae. At the Golgi apparatus newly synthesized proteins are sorted between those transported to the plasma membrane (PM), or the external medium, via the exocytosis or secretory pathway (SEC), and those targeted to the vacuole either through endosomes (vacuolar protein sorting or VPS pathway) or directly (alkaline phosphatase or ALP pathway). Plasma membrane proteins can be internalized by endocytosis (END) and transported to endosomes where they are sorted between those targeted for vacuolar degradation and those redirected to the Golgi (recycling or RCY pathway). Studies in yeast S. cerevisiae allowed the identification of most of the known effectors, protein complexes, and trafficking pathways in eukaryotic cells, and most of them are conserved among eukaryotes.

Btn3 regulates the endosomal sorting function of the yeast Ent3 epsin, an adaptor for SNARE proteins.

Ent3 and Ent5 are yeast epsin N-terminal homology (ENTH) domain-containing proteins involved in protein trafficking between the Golgi and late endosomes. They interact with clathrin, clathrin adaptors at the Golgi (AP-1 and GGA) and different SNAREs (Vti1, Snc1, Pep12 and Syn8) required for vesicular transport at the Golgi and endosomes. To better understand the role of these epsins in membrane trafficking, we performed a protein-protein interaction screen. We identified Btn3 (also known as Tda3), a putative oxidoreductase, as a new partner of both Ent3 and Ent5. Btn3 is a negative regulator of the Batten-disease-linked protein Btn2 involved in the retrieval of specific SNAREs (Vti1, Snc1, Tlg1 and Tlg2) from the late endosome to the Golgi. We show that Btn3 endosomal localization depends on the epsins Ent3 and Ent5. We demonstrated that in btn3Δ mutant cells, endosomal sorting of ubiquitylated cargos and endosomal recycling of the Snc1 SNARE are delayed. We thus propose that Btn3 regulates the sorting function of two adaptors for SNARE proteins, the epsin Ent3 and the Batten-disease-linked protein Btn2.

Study of the plant COPII vesicle coat subunits by functional complementation of yeast Saccharomyces cerevisiae mutants.

The formation and budding of endoplasmic reticulum ER-derived vesicles depends on the COPII coat protein complex that was first identified in yeast Saccharomyces cerevisiae. The ER-associated Sec12 and the Sar1 GTPase initiate the COPII coat formation by recruiting the Sec23-Sec24 heterodimer following the subsequent recruitment of the Sec13-Sec31 heterotetramer. In yeast, there is usually one gene encoding each COPII protein and these proteins are essential for yeast viability, whereas the plant genome encodes multiple isoforms of all COPII subunits. Here, we used a systematic yeast complementation assay to assess the functionality of Arabidopsis thaliana COPII proteins. In this study, the different plant COPII subunits were expressed in their corresponding temperature-sensitive yeast mutant strain to complement their thermosensitivity and secretion phenotypes. Secretion was assessed using two different yeast cargos: the soluble α-factor pheromone and the membranous v-SNARE (vesicle-soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor) Snc1 involved in the fusion of the secretory vesicles with the plasma membrane. This complementation study allowed the identification of functional A. thaliana COPII proteins for the Sec12, Sar1, Sec24 and Sec13 subunits that could represent an active COPII complex in plant cells. Moreover, we found that AtSec12 and AtSec23 were co-immunoprecipitated with AtSar1 in total cell extract of 15 day-old seedlings of A. thaliana. This demonstrates that AtSar1, AtSec12 and AtSec23 can form a protein complex that might represent an active COPII complex in plant cells.

Lsb1 is a negative regulator of las17 dependent actin polymerization involved in endocytosis.

The spatial and temporal regulation of actin polymerization is crucial for various cellular processes. Members of the Wiskott-Aldrich syndrome protein (WASP) family activate the Arp2/3-complex leading to actin polymerization. The yeast Saccharomyces cerevisiae contains only one WASP homolog, Las17, that requires additional factors for its regulation. Lsb1 and Lsb2/Pin3 are two yeast homologous proteins bearing an SH3 domain that were identified as Las17-binding proteins. Lsb2/Pin3 that promotes prion induction was suggested to link this prion formation to the actin cytoskeleton. However, the cellular role of Lsb1 and the molecular function of both Lsb1 and Lsb2 remain unknown. In this study, we show that Lsb1 and/or Lsb2 full-length proteins inhibit Las17-mediated actin polymerization in vitro, Lsb2 being a less potent inhibitor of Las17 activity compared to Lsb1. Addition of Lsb1 or Lsb2 to the corresponding full-length Lsb1/2 further inhibits Las17 activity. Lsb1 and Lsb2 form homo- and hetero-oligomeric complexes suggesting that these two proteins could regulate Las17 activity via dimerization or cooperative binding. In vivo, overexpressed Lsb1 and Lsb2 proteins cluster Las17-CFP in few cytoplasmic punctate structures that are also positive for other Arp2/3-dependent actin polymerization effectors like Sla1 or Abp1. But, only Lsb1 overexpression blocks the internalization step of receptor-mediated endocytosis. This shows a specific function of Lsb1 in endocytosis.

Evolutionary analysis of the ENTH/ANTH/VHS protein superfamily reveals a coevolution between membrane trafficking and metabolism.

BACKGROUND: Membrane trafficking involves the complex regulation of proteins and lipids intracellular localization and is required for metabolic uptake, cell growth and development. Different trafficking pathways passing through the endosomes are coordinated by the ENTH/ANTH/VHS adaptor protein superfamily. The endosomes are crucial for eukaryotes since the acquisition of the endomembrane system was a central process in eukaryogenesis. RESULTS: Our in silico analysis of this ENTH/ANTH/VHS superfamily, consisting of proteins gathered from 84 complete genomes representative of the different eukaryotic taxa, revealed that genomic distribution of this superfamily allows to discriminate Fungi and Metazoa from Plantae and Protists. Next, in a four way genome wide comparison, we showed that this discriminative feature is observed not only for other membrane trafficking effectors, but also for proteins involved in metabolism and in cytokinesis, suggesting that metabolism, cytokinesis and intracellular trafficking pathways co-evolved. Moreover, some of the proteins identified were implicated in multiple functions, in either trafficking and metabolism or trafficking and cytokinesis, suggesting that membrane trafficking is central to this co-evolution process. CONCLUSIONS: Our study suggests that membrane trafficking and compartmentalization were not only key features for the emergence of eukaryotic cells but also drove the separation of the eukaryotes in the different taxa.

Rtn1p is involved in structuring the cortical endoplasmic reticulum.

The endoplasmic reticulum (ER) contains both cisternal and reticular elements in one contiguous structure. We identified rtn1Delta in a systematic screen for yeast mutants with altered ER morphology. The ER in rtn1Delta cells is predominantly cisternal rather than reticular, yet the net surface area of ER is not significantly changed. Rtn1-green fluorescent protein (GFP) associates with the reticular ER at the cell cortex and with the tubules that connect the cortical ER to the nuclear envelope, but not with the nuclear envelope itself. Rtn1p overexpression also results in an altered ER structure. Rtn proteins are found on the ER in a wide range of eukaryotes and are defined by two membrane-spanning domains flanking a conserved hydrophilic loop. Our results suggest that Rtn proteins may direct the formation of reticulated ER. We independently identified Rtn1p in a proteomic screen for proteins associated with the exocyst vesicle tethering complex. The conserved hydophilic loop of Rtn1p binds to the exocyst subunit Sec6p. Overexpression of this loop results in a modest accumulation of secretory vesicles, suggesting impaired exocyst function. The interaction of Rtn1p with the exocyst at the bud tip may trigger the formation of a cortical ER network in yeast buds.

Functional specialization within a vesicle tethering complex: bypass of a subset of exocyst deletion mutants by Sec1p or Sec4p.

The exocyst is an octameric protein complex required to tether secretory vesicles to exocytic sites and to retain ER tubules at the apical tip of budded cells. Unlike the other five exocyst genes, SEC3, SEC5, and EXO70 are not essential for growth or secretion when either the upstream activator rab, Sec4p, or the downstream SNARE-binding component, Sec1p, are overproduced. Analysis of the suppressed sec3Delta, sec5Delta, and exo70Delta strains demonstrates that the corresponding proteins confer differential effects on vesicle targeting and ER inheritance. Sec3p and Sec5p are more critical than Exo70p for ER inheritance. Although nonessential under these conditions, Sec3p, Sec5p, and Exo70p are still important for tethering, as in their absence the exocyst is only partially assembled. Sec1p overproduction results in increased SNARE complex levels, indicating a role in assembly or stabilization of SNARE complexes. Furthermore, a fraction of Sec1p can be coprecipitated with the exoycst. Our results suggest that Sec1p couples exocyst-mediated vesicle tethering with SNARE-mediated docking and fusion.