Yeast Secretory Pathway Research Articles

A Yeast Modular Cloning (MoClo) Toolkit Expansion for Optimization of Heterologous Protein Secretion and Surface Display in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is an attractive host for the expression of secreted proteins in a biotechnology context. Unfortunately, many heterologous proteins fail to enter, or efficiently progress through, the secretory pathway, resulting in poor yields. Similarly, yeast surface display has become a widely used technique in protein engineering but achieving sufficient levels of surface expression of recombinant proteins is often challenging. Signal peptides (SPs) and translational fusion partners (TFPs) can be used to direct heterologous proteins through the yeast secretory pathway, however, selection of the optimal secretion promoting sequence is largely a process of trial and error. The yeast modular cloning (MoClo) toolkit utilizes type IIS restriction enzymes to facilitate an efficient assembly of expression vectors from standardized parts. We have expanded this toolkit to enable the efficient incorporation of a panel of 16 well-characterized SPs and TFPs and five surface display anchor proteins into S. cerevisiae expression cassettes. The secretion promoting signals are validated by using five different proteins of interest. Comparison of intracellular and secreted protein levels reveals the optimal secretion promoting sequence for each individual protein. Large, protein of interest-specific variations in secretion efficiency are observed. SP sequences are also used with the five surface display anchors, and the combination of SP and anchor protein proves critical for efficient surface display. These observations highlight the value of the described panel of MoClo compatible parts to allow facile screening of SPs and TFPs and anchor proteins for optimal secretion and/or surface display of a given protein of interest in S. cerevisiae.

High-resolution secretory timeline from vesicle formation at the Golgi to fusion at the plasma membrane in S. cerevisiae.

Most of the components in the yeast secretory pathway have been studied, yet a high-resolution temporal timeline of their participation is lacking. Here, we define the order of acquisition, lifetime, and release of critical components involved in late secretion from the Golgi to the plasma membrane. Of particular interest is the timing of the many reported effectors of the secretory vesicle Rab protein Sec4, including the myosin-V Myo2, the exocyst complex, the lgl homolog Sro7, and the small yeast-specific protein Mso1. At the trans-Golgi network (TGN) Sec4's GEF, Sec2, is recruited to Ypt31-positive compartments, quickly followed by Sec4 and Myo2 and vesicle formation. While transported to the bud tip, the entire exocyst complex, including Sec3, is assembled on to the vesicle. Before fusion, vesicles tether for 5 s, during which the vesicle retains the exocyst complex and stimulates lateral recruitment of Rho3 on the plasma membrane. Sec2 and Myo2 are rapidly lost, followed by recruitment of cytosolic Sro7, and finally the SM protein Sec1, which appears for just 2 s prior to fusion. Perturbation experiments reveal an ordered and robust series of events during tethering that provide insights into the function of Sec4 and effector exchange.

Pushing and pulling proteins into the yeast secretory pathway enhances recombinant protein secretion

Yeasts and especially Pichia pastoris (syn Komagataella spp.) are popular microbial expression systems for the production of recombinant proteins. One of the key advantages of yeast host systems is their ability to secrete the recombinant protein into the culture media. However, secretion of some recombinant proteins is less efficient. These proteins include antibody fragments such as Fabs or scFvs. We have recently identified translocation of nascent Fab fragments from the cytosol into the endoplasmic reticulum (ER) as one major bottleneck. Conceptually, this bottleneck requires engineering to increase the flux of recombinant proteins at the translocation step by pushing on the cytosolic side and pulling on the ER side. This engineering strategy is well-known in the field of metabolic engineering. To apply the push-and-pull strategy to recombinant protein secretion, we chose to modulate the cytosolic and ER Hsp70 cycles, which have a key impact on the translocation process. After identifying the relevant candidate factors of the Hsp70 cycles, we combined the push-and-pull factors in a single strain and achieved synergistic effects for antibody fragment secretion. With this concept we were able to successfully engineer strains and improve protein secretion up to 5-fold for different model protein classes. Overall, titers of more than 1.3 g/L Fab and scFv were reached in bioreactor cultivations.

Synthetic pro-peptide design to enhance the secretion of heterologous proteins by Saccharomyces cerevisiae.

Heterologous protein production in Saccharomyces cerevisiae is a useful and effective strategy with many advantages, including the secretion of proteins that require posttranslational processing. However, heterologous proteins in S. cerevisiae are often secreted at comparatively low levels. To improve the production of the heterologous protein, human granulocyte colony‐stimulating factor (hG‐CSF) in S. cerevisiae, a secretion‐enhancing peptide cassette including an hIL‐1β‐derived propeptide, was added and used as a secretion enhancer to alleviate specific bottlenecks in the yeast secretory pathway. The effects of three key parameters—N‐glycosylation, net negative charge balance, and glycine‐rich flexible linker—were investigated in batch cultures of S. cerevisiae. Using a three‐stage design involving screening, selection, and optimization, the production and secretion of hG‐CSF by S. cerevisiae were significantly increased. The amount of extracellular mature hG‐CSF produced by the optimized propeptide after the final stage increased by 190% compared to that of the original propeptide. Although hG‐CSF was used as the model protein in the current study, this strategy applies to the enhanced production of other heterologous proteins, using S. cerevisiae as the host.

How presence of a signal peptide affects human galectins-1 and -4: Clues to explain common absence of a leader sequence among adhesion/growth-regulatory galectins

BackgroundGalectins are multifunctional effectors, which all share absence of a signal sequence. It is not clear why galectins belong to the small set of proteins, which avoid the classical export route. MethodsProducts of recombinant galectin expression in P. pastoris were analyzed by haemagglutination, gel filtration and electrophoresis and lectin blotting as well as mass spectrometry on the level of tryptic peptides and purified glycopeptides(s). Density gradient centrifugation and confocal laser scanning microscopy facilitated localization in transfected human and rat cells, proliferation assays determined activity as growth mediator. ResultsDirecting galectin-1 to the classical secretory pathway in yeast produces N-glycosylated protein that is active. It cofractionates and -localizes with calnexin in human cells, only Gal-4 is secreted. Presence of N-glycan(s) reduces affinity of cell binding and growth regulation by Gal-1. ConclusionsFolding and activity of a galectin are maintained in signal-peptide-directed routing, N-glycosylation occurs. This pathway would deplete cytoplasm and nucleus of galectin, presence of N-glycans appears to interfere with lattice formation. General significanceAvailability of glycosylated galectins facilitates functional assays to contribute to explain why galectins invariably avoid classical routing for export.

Cucumber Golgi protein CsMTP5 forms a Zn-transporting heterodimer with high molecular mass protein CsMTP12

Heterodimeric complexes formed by members of the cation facilitator (CDF) family catalyse the import of Zn into the secretory pathway of yeast and vertebrate cells. Orthologous proteins AtMTP5 and AtMTP12 from Arabidopsis have also been shown to form a heterodimeric complex at the Golgi compartment of plant cells that possibly transport Zn. In this study we show that cucumber proteins CsMTP5 and CsMTP12 form a functional heterodimer that is involved in the loading of Zn into the ER lumen under low Zn, and not in the detoxification of yeast from Zn excess through vesicle-mediated exocytosis. Using specific antibodies, we demonstrate that CsMTP5 is localized at the Golgi compartment of cucumber cells and is markedly up-regulated upon Zn deficiency. The level of CsMTP5 transcript in cucumber is also significantly elevated in Zn-limiting conditions, whereas the expression of CsMTP12 is independent of the availability of Zn. Therefore we propose that the cucumber heterodimeric complex CsMTP5-CsMTP12 functions to deliver Zn to Zn-dependent proteins of the Golgi compartment and is regulated by zinc at the level of CsMTP5 transcription.

Rewiring a Rab regulatory network reveals a possible inhibitory role for the vesicle tether, Uso1

Ypt1 and Sec4 are essential Rab GTPases that control the early and late stages of the yeast secretory pathway, respectively. A chimera consisting of Ypt1 with the switch I domain of Sec4, Ypt1-SW1Sec4, is efficiently activated in vitro by the Sec4 exchange factor, Sec2. This should lead to its ectopic activation in vivo and thereby disrupt membrane traffic. Nonetheless early studies found that yeast expressing Ypt1-SW1Sec4 as the sole copy of YPT1 exhibit no growth defect. To resolve this conundrum, we have analyzed yeast expressing various levels of Ypt1-SW1Sec4 We show that even normal expression of Ypt1-SW1Sec4 leads to kinetic transport defects at a late stage of the pathway, with secretory vesicles accumulating near exocytic sites. Higher levels are toxic. Toxicity is suppressed by truncation of Uso1, a vesicle tether required for endoplasmic reticulum-Golgi traffic. The globular head of Uso1 binds to Ypt1 and its coiled-coil tail binds to the Golgi-associated SNARE, Sed5. We propose that when Uso1 is inappropriately recruited to secretory vesicles by Ypt1-SW1Sec4, the extended coiled-coil tail blocks docking to the plasma membrane. This putative inhibitory function could serve to increase the fidelity of vesicle docking.

Moderate Expression of SEC16 Increases Protein Secretion by Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is widely used to produce biopharmaceutical proteins. However, the limited capacity of the secretory pathway may reduce its productivity. Here, we increased the secretion of a heterologous α-amylase, a model protein used for studying the protein secretory pathway in yeast, by moderately overexpressing SEC16, which is involved in protein translocation from the endoplasmic reticulum to the Golgi apparatus. The moderate overexpression of SEC16 increased α-amylase secretion by generating more endoplasmic reticulum exit sites. The production of reactive oxygen species resulting from the heterologous α-amylase production was reduced. A genome-wide expression analysis indicated decreased endoplasmic reticulum stress in the strain that moderately overexpressed SEC16, which was consistent with a decreased volume of the endoplasmic reticulum. Additionally, fewer mitochondria were observed. Finally, the moderate overexpression of SEC16 was shown to improve the secretion of two other recombinant proteins, Trichoderma reesei endoglucanase I and Rhizopus oryzae glucan-1,4-α-glucosidase, indicating that this mechanism is of general relevance.IMPORTANCE There is an increasing demand for recombinant proteins to be used as enzymes and pharmaceuticals. The yeast Saccharomyces cerevisiae is a cell factory that is widely used to produce recombinant proteins. Our study revealed that moderate overexpression of SEC16 increased recombinant protein secretion in S. cerevisiae This new strategy can be combined with other targets to engineer cell factories to efficiently produce protein in the future.

Network reconstruction of the mouse secretory pathway applied on CHO cell transcriptome data

BackgroundProtein secretion is one of the most important processes in eukaryotes. It is based on a highly complex machinery involving numerous proteins in several cellular compartments. The elucidation of the cell biology of the secretory machinery is of great importance, as it drives protein expression for biopharmaceutical industry, a 140 billion USD global market. However, the complexity of secretory process is difficult to describe using a simple reductionist approach, and therefore a promising avenue is to employ the tools of systems biology.ResultsOn the basis of manual curation of the literature on the yeast, human, and mouse secretory pathway, we have compiled a comprehensive catalogue of characterized proteins with functional annotation and their interconnectivity. Thus we have established the most elaborate reconstruction (RECON) of the functional secretion pathway network to date, counting 801 different components in mouse. By employing our mouse RECON to the CHO-K1 genome in a comparative genomic approach, we could reconstruct the protein secretory pathway of CHO cells counting 764 CHO components. This RECON furthermore facilitated the development of three alternative methods to study protein secretion through graphical visualizations of omics data. We have demonstrated the use of these methods to identify potential new and known targets for engineering improved growth and IgG production, as well as the general observation that CHO cells seem to have less strict transcriptional regulation of protein secretion than healthy mouse cells.ConclusionsThe RECON of the secretory pathway represents a strong tool for interpretation of data related to protein secretion as illustrated with transcriptomic data of Chinese Hamster Ovary (CHO) cells, the main platform for mammalian protein production.

Engineering vesicle trafficking improves the extracellular activity and surface display efficiency of cellulases in Saccharomyces cerevisiae

BackgroundCellulase expression via extracellular secretion or surface display in Saccharomyces cerevisiae is one of the most frequently used strategies for a consolidated bioprocess (CBP) of cellulosic ethanol production. However, the inefficiency of the yeast secretory pathway often results in low production of heterologous proteins, which largely limits cellulase secretion or display.ResultsIn this study, the components of the vesicle trafficking from the endoplasmic reticulum (ER) to the Golgi and from the Golgi to the plasma membrane, involved in vesicle budding, tethering and fusion, were over-expressed in Clostridium thermocellum endoglucanase (CelA)- and Sacchromycopsis fibuligera β-glucosidase (BGL1)-secreting or -displaying strains. Engineering the targeted components in the ER to Golgi vesicle trafficking, including Sec12p, Sec13p, Erv25p and Bos1p, enhanced the extracellular activity of CelA. However, only Sec13p over-expression increased BGL1 secretion. By contrast, over-expression of the components in the Golgi to plasma membrane vesicle trafficking, including Sso1p, Snc2p, Sec1p, Exo70p, Ypt32p and Sec4p, showed better performance in increasing BGL1 secretion compared to CelA secretion, and the over-expression of these components all increased BGL1 extracellular activity. These results revealed that various cellulases showed different limitations in protein transport, and engineering vesicle trafficking has protein-specific effects. Importantly, we found that engineering the above vesicle trafficking components, particularly from the ER to the Golgi, also improved the display efficiency of CelA and BGL1 when a-agglutinin was used as surface display system. Further analyses illustrated that the display efficiency of a-agglutinin was increased by engineering vesicle trafficking, and the trend was consistent with displayed CelA and BGL1. These results indicated that fusion with a-agglutinin may affect the proteins’ properties and alter the rate-limiting step in the vesicle trafficking.ConclusionsWe have demonstrated, for the first time, engineering vesicle trafficking from the ER to the Golgi and from the Golgi to the plasma membrane can enhance the protein display efficiency. We also found that different heterologous proteins had specific limitations in vesicle trafficking pathway and that engineering the vesicle trafficking resulted in a protein-specific effect. These results provide a new strategy to improve the extracellular secretion and surface display of cellulases in S. cerevisiae.

Profiling Protease Specificity: Combining Yeast ER Sequestration Screening (YESS) with Next Generation Sequencing.

An enzyme engineering technology involving yeast endoplasmic reticulum (ER) sequestration screening (YESS) has been recently developed. Here, a new method is established, in which the YESS platform is combined with NextGen sequencing (NGS) to enable a comprehensive survey of protease specificity. In this approach, a combinatorial substrate library is targeted to the yeast ER and transported through the secretory pathway, interacting with any protease(s) residing in the ER. Multicolor FACS screening is used to isolate cells labeled with fluorophore-conjugated antibodies, followed by NGS to profile the cleaved substrates. The YESS-NGS method was successfully applied to profile the sequence specificity of the wild-type and an engineered variant of the tobacco etch mosaic virus protease. Proteolysis in the yeast secretory pathway was also mapped for the first time in vivo revealing a major cleavage pattern of Ali/Leu-X-Lys/Arg-Arg. Here Ali is any small aliphatic residue, but especially Leu. This pattern was verified to be due to the well-known endogenous protease Kex2 after comparison to a newly generated Kex2 knockout strain as well as cleavage of peptides with recombinant Kex2 in vitro. This information is particularly important for those using yeast display technology, as library members with Ali/Leu-X-Lys/Arg-Arg patterns are likely being removed from screens via Kex2 cleavage without the researcher's knowledge.

The conundrum of UDP-Glc entrance into the yeast ER lumen.

UDP-Glc entrance into the endoplasmic reticulum (ER) of eukaryotic cells is a key step in the quality control of glycoprotein folding, a mechanism requiring transfer of a Glc residue from the nucleotide sugar (NS) to glycoprotein folding intermediates by the UDP-Glc:glycoprotein glucosyltransferase (UGGT). According to a bioinformatics search there are only eight genes in the Schizosaccharomyces pombe genome belonging to the three Pfam families to which all known nucleotide-sugar transporters (NSTs) of the secretory pathway belong. The protein products of two of them (hut1+ and yea4+) localize to the ER, those of genes gms1+, vrg4+, pet1+, pet2+ and pet3+ to the Golgi, whereas that of gms2+ has an unknown location. Here we demonstrate that (1) Δhut1 and Δgpt1 (UGGT null) mutants share several phenotypic features; (2) Δhut1 mutants show a 50% reduction in UDP-Glc transport into ER-derived membranes; (3) in vivo UDP-Glc ER entrance occurred in Δhut1Δyea4Δgms2 mutants and in cells in which Δhut1 disruption was combined with that of each of four of the genes encoding Golgi-located proteins. Therefore, disruption of all genes whose products localize to the ER or have an unknown location did not obliterate UDP-Glc ER entrance. We conclude that the hut1+ gene product is involved in UDP-Glc entrance into the ER, but that at least another as yet unknown NST displaying an unconventional sequence operates in the yeast secretory pathway. This conclusion agrees with our previous results showing that UDP-Glc entrance into the yeast ER does not follow the classical NST antiport mechanism.

The casein kinases Yck1p and Yck2p act in the secretory pathway, in part, by regulating the Rab exchange factor Sec2p.

Sec2p is a guanine nucleotide exchange factor that activates Sec4p, the final Rab GTPase of the yeast secretory pathway. Sec2p is recruited to secretory vesicles by the upstream Rab Ypt32p acting in concert with phosphatidylinositol-4-phosphate (PI(4)P). Sec2p also binds to the Sec4p effector Sec15p, yet Ypt32p and Sec15p compete against each other for binding to Sec2p. We report here that the redundant casein kinases Yck1p and Yck2p phosphorylate sites within the Ypt32p/Sec15p binding region and in doing so promote binding to Sec15p and inhibit binding to Ypt32p. We show that Yck2p binds to the autoinhibitory domain of Sec2p, adjacent to the PI(4)P binding site, and that addition of PI(4)P inhibits Sec2p phosphorylation by Yck2p. Loss of Yck1p and Yck2p function leads to accumulation of an intracellular pool of the secreted glucanase Bgl2p, as well as to accumulation of Golgi-related structures in the cytoplasm. We propose that Sec2p is phosphorylated after it has been recruited to secretory vesicles and the level of PI(4)P has been reduced. This promotes Sec2p function by stimulating its interaction with Sec15p. Finally, Sec2p is dephosphorylated very late in the exocytic reaction to facilitate recycling.

Secretion of a foreign protein from budding yeasts is enhanced by cotranslational translocation and by suppression of vacuolar targeting.

BackgroundBudding yeasts are often used to secrete foreign proteins, but the efficiency is variable. To identify roadblocks in the yeast secretory pathway, we used a monomeric superfolder GFP (msGFP) as a visual tracer in Saccharomyces cerevisiae and Pichia pastoris.ResultsOne roadblock for msGFP secretion is translocation into the ER. Foreign proteins are typically fused to the bipartite α-factor secretion signal, which consists of the signal sequence followed by the pro region. The α-factor signal sequence directs posttranslational translocation. For msGFP, posttranslational translocation is inefficient with the α-factor signal sequence alone but is stimulated by the pro region. This requirement for the pro region can be bypassed by using the Ost1 signal sequence, which has been shown to direct cotranslational translocation. A hybrid secretion signal consisting of the Ost1 signal sequence followed by the α-factor pro region drives efficient translocation followed by rapid ER export. A second roadblock for msGFP secretion in S. cerevisiae occurs during exit from the Golgi, when some of the msGFP molecules are diverted to the vacuole. Deletion of the sorting receptor Vps10 prevents vacuolar targeting of msGFP at the expense of missorting vacuolar hydrolases such as carboxypeptidase Y (CPY) to the culture medium. However, a truncation of Vps10 blocks vacuolar targeting of msGFP while permitting CPY to be sorted normally.ConclusionsWith budding yeasts, if the secretion or processing of a foreign protein is poor, we recommend two options. First, use the Ost1 signal sequence to achieve efficient entry into the secretory pathway while avoiding the processing issues associated with the α-factor pro region. Second, truncate Vps10 to suppress diversion to the vacuole. These insights obtained with msGFP highlight the value of applying cell biological methods to study yeast secretion.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-014-0125-0) contains supplementary material, which is available to authorized users.

The complexity of vesicle transport factors in plants examined by orthology search.

Vesicle transport is a central process to ensure protein and lipid distribution in eukaryotic cells. The current knowledge on the molecular components and mechanisms of this process is majorly based on studies in Saccharomyces cerevisiae and Arabidopsis thaliana, which revealed 240 different proteinaceous factors either experimentally proven or predicted to be involved in vesicle transport. In here, we performed an orthologue search using two different algorithms to identify the components of the secretory pathway in yeast and 14 plant genomes by using the ‘core-set’ of 240 factors as bait. We identified 4021 orthologues and (co-)orthologues in the discussed plant species accounting for components of COP-II, COP-I, Clathrin Coated Vesicles, Retromers and ESCRTs, Rab GTPases, Tethering factors and SNAREs. In plants, we observed a significantly higher number of (co-)orthologues than yeast, while only 8 tethering factors from yeast seem to be absent in the analyzed plant genomes. To link the identified (co-)orthologues to vesicle transport, the domain architecture of the proteins from yeast, genetic model plant A. thaliana and agriculturally relevant crop Solanum lycopersicum has been inspected. For the orthologous groups containing (co-)orthologues from yeast, A. thaliana and S. lycopersicum, we observed the same domain architecture for 79% (416/527) of the (co-)orthologues, which documents a very high conservation of this process. Further, publically available tissue-specific expression profiles for a subset of (co-)orthologues found in A. thaliana and S. lycopersicum suggest that some (co-)orthologues are involved in tissue-specific functions. Inspection of localization of the (co-)orthologues based on available proteome data or localization predictions lead to the assignment of plastid- as well as mitochondrial localized (co-)orthologues of vesicle transport factors and the relevance of this is discussed.

Secretion of a foreign protein from budding yeasts is enhanced by cotranslational translocation and by suppression of vacuolar targeting

Budding yeasts are often used to secrete foreign proteins, but the efficiency is variable. To identify roadblocks in the yeast secretory pathway, we used a monomeric superfolder GFP (msGFP) as a visual tracer in Saccharomyces cerevisiae and Pichia pastoris. One roadblock for msGFP secretion is translocation into the ER. Foreign proteins are typically fused to the bipartite α-factor secretion signal, which consists of the signal sequence followed by the pro region. The α-factor signal sequence directs posttranslational translocation. For msGFP, posttranslational translocation is inefficient with the α-factor signal sequence alone but is stimulated by the pro region. This requirement for the pro region can be bypassed by using the Ost1 signal sequence, which has been shown to direct cotranslational translocation. A hybrid secretion signal consisting of the Ost1 signal sequence followed by the α-factor pro region drives efficient translocation followed by rapid ER export. A second roadblock for msGFP secretion in S. cerevisiae occurs during exit from the Golgi, when some of the msGFP molecules are diverted to the vacuole. Deletion of the sorting receptor Vps10 prevents vacuolar targeting of msGFP at the expense of missorting vacuolar hydrolases such as carboxypeptidase Y (CPY) to the culture medium. However, a truncation of Vps10 blocks vacuolar targeting of msGFP while permitting CPY to be sorted normally. With budding yeasts, if the secretion or processing of a foreign protein is poor, we recommend two options. First, use the Ost1 signal sequence to achieve efficient entry into the secretory pathway while avoiding the processing issues associated with the α-factor pro region. Second, truncate Vps10 to suppress diversion to the vacuole. These insights obtained with msGFP highlight the value of applying cell biological methods to study yeast secretion.

Profile of Peter Novick

There was a time in graduate school when Peter Novick wasn’t sure if his research would lead anywhere. As a graduate student in cell biologist Randy Schekman’s laboratory at the University of California at Berkeley, Novick had been using a genetic approach to understand the yeast secretory pathway, responsible for moving proteins out of the cell. Now a professor at the University of California, San Diego, Novick says it was only when he identified a mutant in which vesicles piled up inside the cell, showing that it had a defective secretory pathway, that he knew he had made a breakthrough. “That convinced me. Before then I wasn’t sure if I had a thesis project, afterwards it was pretty clear I did,” he says. Peter Novick. It turned out that Novick had a lot more than a thesis project. His graduate work paved the way for Schekman’s detailed analysis of the secretory pathway, work for which Schekman shared the 2013 Nobel Prize in Physiology or Medicine. “Peter Novick was instrumental in starting our genetic approach to the secretory pathway in yeast,” says Schekman. “Already as a first year graduate student, he showed great skill and devised the screen and a selection procedure that allowed us to isolate the first mutant, sec1-1, and within another year, mutations representing 23 genes that defined the major stages in the secretory pathway,” he says. In eukaryotic cells, such as yeast and higher organisms, the secretory pathway transports proteins from the endoplasmic reticulum (ER), an organelle where the proteins are synthesized, to the Golgi apparatus, a cellular compartment that serves as a sorting station. From the Golgi, the proteins then move to the cells’ plasma membrane. The latter step involves transporting the proteins in vesicles that fuse with the plasma membrane and release proteins to the outside of the cell. The regulation of such intracellular trafficking is relevant to many human diseases, including cancer and diabetes. Understanding how yeast secretes proteins has also been used in biotechnology to produce proteins such as insulin. Novick says his graduate work led to his continued interest in teasing apart the many components of the secretory pathway. Using a combination of yeast genetics and cell biology, he has spent his career investigating the tightly regulated mechanisms involved in intracellular transport. For his contributions to our understanding of this fundamental physiological process, Novick was elected to the National Academy of Sciences in 2013. “In his own lab, first at Yale and now at UC San Diego, Peter launched a brilliant independent career with the discovery that a protein called Sec4 encodes a small GTP-binding protein, the first of three dozen so-called Rab proteins that we now know control the targeting of transport vesicles to all the many destinations in the cell,” says Schekman. “On the strength of this work and much more in subsequent years, he was elected to the National Academy of Sciences, an honor that was in my opinion long overdue,” he says.

Characterization of the Pichia pastoris Protein-O-mannosyltransferase Gene Family

The methylotrophic yeast, Pichia pastoris , is an important organism used for the production of therapeutic proteins. However, the presence of fungal-like glycans, either N-linked or O-linked, can elicit an immune response or enable the expressed protein to bind to mannose receptors, thus reducing their efficacy. Previously we have reported the elimination of β-linked glycans in this organism. In the current report we have focused on reducing the O-linked mannose content of proteins produced in P . pastoris , thereby reducing the potential to bind to mannose receptors. The initial step in the synthesis of O-linked glycans in P . pastoris is the transfer of mannose from dolichol-phosphomannose to a target protein in the yeast secretory pathway by members of the protein-O-mannosyltransferase (PMT) family. In this report we identify and characterize the members of the P . pastoris PMT family. Like Candida albicans, P . pastoris has five PMT genes. Based on sequence homology, these PMTs can be grouped into three sub-families, with both PMT1 and PMT2 sub-families possessing two members each (PMT1 and PMT5, and PMT2 and PMT6, respectively). The remaining sub-family, PMT4, has only one member (PMT4). Through gene knockouts we show that PMT1 and PMT2 each play a significant role in O-glycosylation. Both, by gene knockouts and the use of Pmt inhibitors we were able to significantly reduce not only the degree of O-mannosylation, but also the chain-length of these glycans. Taken together, this reduction of O-glycosylation represents an important step forward in developing the P . pastoris platform as a suitable system for the production of therapeutic glycoproteins.

Impaired Manganese Metabolism Causes Mitotic Misregulation

Manganese is an essential trace element, whose intracellular levels need to be carefully regulated. Mn(2+) acts as a cofactor for many enzymes and excess of Mn(2+) is toxic. Alterations in Mn(2+) homeostasis affect metabolic functions and mutations in the human Mn(2+)/Ca(2+) transporter ATP2C1 have been linked to Hailey-Hailey disease. By deletion of the yeast orthologue PMR1 we have studied the impact of Mn(2+) on cell cycle progression and show that an excess of cytosolic Mn(2+) alters S-phase transit, induces transcriptional up-regulation of cell cycle regulators, bypasses the need for S-phase cell cycle checkpoints and predisposes to genomic instability. On the other hand, we find that depletion of the Golgi Mn(2+) pool requires a functional morphology checkpoint to avoid the formation of polyploid cells.

Molecular basis of the tarantula toxin jingzhaotoxin‐III (β‐TRTX‐Cj1α) interacting with voltage sensors in sodium channel subtype Nav1.5

With conserved structural scaffold and divergent electrophysiological functions, animal toxins are considered powerful tools for investigating the basic structure-function relationship of voltage-gated sodium channels. Jingzhaotoxin-III (β-TRTX-Cj1α) is a unique sodium channel gating modifier from the tarantula Chilobrachys jingzhao, because the toxin can selectively inhibit the activation of cardiac sodium channel but not neuronal subtypes. However, the molecular basis of JZTX-III interaction with sodium channels remains unknown. In this study, we showed that JZTX-III was efficiently expressed by the secretory pathway in yeast. Alanine-scanning analysis indicated that 2 acidic residues (Asp1, Glu3) and an exposed hydrophobic patch, formed by 4 Trp residues (residues 8, 9, 28 and 30), play important roles in the binding of JZTX-III to Nav1.5. JZTX-III docked to the Nav1.5 DIIS3-S4 linker. Mutations S799A, R800A, and L804A could additively reduce toxin sensitivity of Nav1.5. We also demonstrated that the unique Arg800, not emerging in other sodium channel subtypes, is responsible for JZTX-III selectively interacting with Nav1.5. The reverse mutation D816R in Nav1.7 greatly increased the sensitivity of the neuronal subtype to JZTX-III. Conversely, the mutation R800D in Nav1.5 decreased JZTX-III's IC₅₀ by 72-fold. Therefore, our results indicated that JZTX-III is a site 4 toxin, but does not possess the same critical residues on sodium channels as other site 4 toxins. Our data also revealed the underlying mechanism for JZTX-III to be highly specific for the cardiac sodium channel.