An improved secretion signal enhances the secretion of model proteins from Pichia pastoris

Background: proteins can be secreted from a host organism with the aid of N-terminal secretion signals. The budding yeast Pichia pastoris (Komagataella sp.) is widely employed to secrete proteins of academic and industrial interest. For this yeast, the most commonly used secretion signal is the N-terminal portion of pre-pro-α-factor from Saccharomyces cerevisiae. However, this secretion signal promotes posttranslational translocation into the endoplasmic reticulum (ER), so proteins that can fold in the cytosol may be inefficiently translocated and thus poorly secreted. In addition, if a protein self-associates, the α-factor pro region can potentially cause aggregation, thereby hampering export from the ER. This study addresses both limitations of the pre-pro-α-factor secretion signal. - Results: we engineered a hybrid secretion signal consisting of the S. cerevisiae Ost1 signal sequence, which promotes cotranslational translocation into the ER, followed by the α-factor pro region. Secretion and intracellular localization were assessed using as a model protein the tetrameric red fluorescent protein E2-Crimson. When paired with the α-factor pro region, the Ost1 signal sequence yielded much more efficient secretion than the α-factor signal sequence. Moreover, an allelic variant of the α-factor pro region reduced aggregation of the E2-Crimson construct in the ER. The resulting improved secretion signal enhanced secretion of E2-Crimson up to 20-fold compared to the levels obtained with the original α-factor secretion signal. Similar findings were obtained with the lipase BTL2, which exhibited 10-fold enhanced secretion with the improved secretion signal. - Conclusions: the improved secretion signal confers dramatic benefits for the secretion of certain proteins from P. pastoris. These benefits are likely to be most evident for proteins that can fold in the cytosol and for oligomeric proteins

ESCargo: a regulatable fluorescent secretory cargo for diverse model organisms

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Casler, J. C., Zajac, A. L., Valbuena, F. M., Sparvoli, D., Jeyifous, O., Turkewitz, A. P., Horne-Badovinac, S., Green, W. N., & Glick, B. S. ESCargo: a regulatable fluorescent secretory cargo for diverse model organisms. Molecular Biology of the Cell, (2020): mbcE20090591, doi:10.1091/mbc.E20-09-0591.Membrane traffic can be studied by imaging a cargo protein as it transits the secretory pathway. The best tools for this purpose initially block export of the secretory cargo from the endoplasmic reticulum (ER), and then release the block to generate a cargo wave. However, previously developed regulatable secretory cargoes are often tricky to use or specific for a single model organism. To overcome these hurdles for budding yeast, we recently optimized an artificial fluorescent secretory protein that exits the ER with the aid of the Erv29 cargo receptor, which is homologous to mammalian Surf4. The fluorescentsecretory protein forms aggregates in the ER lumen and can be rapidly disaggregated by addition of a ligand to generate a nearly synchronized cargo wave. Here we term this regulatable secretory proteinESCargo (Erv29/Surf4-dependent Secretory Cargo) and demonstrate its utility not only in yeast cells, but also in cultured mammalian cells, Drosophila cells, and the ciliate Tetrahymena thermophila. Kinetic studies indicate that rapid export from the ER requires recognition by Erv29/Surf4. By choosing an appropriate ER signal sequence and expression vector, this simple technology can likely be used withmany model organisms.This work was supported by NIH grant R01 GM104010 to BSG, by NIH grant R01 GM105783 to APT, by NIH grant R01 GM136961 and American Cancer Society grant RSG-14-176 to SHB, and by NIH grant R01 DA044760 to WNG. JCC was supported by NIH training grant T32 GM007183. AZ was supported by American Heart Association fellowship 16POST2726018 and American Cancer Society fellowship 132123-PF-18-025-01-CSM. Thanks for assistance with fluorescence microscopy to Vytas Bindokas and Christine Labno at the Integrated Microscopy Core Facility, which is supported by the NIH-funded Cancer Center Support Grant P30 CA014599. The pUASt-ManII-eGFP plasmid was a gift from Bing Ye, and the Ubi-Gal4 plasmid was a gift from Rick Fehon.2020-12-2

Activity-dependent Golgi satellite formation in dendrites reshapes the neuronal surface glycoproteome

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Govind, A. P., Jeyifous, O., Russell, T. A., Yi, Z., Weigel, A., Ramaprasad, A., Newell, L., Ramos, W., Valbuena, F. M., Casler, J. C., Yan, J.-Z., Glick, B. S., Swanson, G. T., Lippincott-Schwartz, J., & Green, W. N. Activity-dependent Golgi satellite formation in dendrites reshapes the neuronal surface glycoproteome. Elife, 10, (2021): e68910, https://doi.org/10.7554/eLife.68910.Activity-driven changes in the neuronal surface glycoproteome are known to occur with synapse formation, plasticity, and related diseases, but their mechanistic basis and significance are unclear. Here, we observed that N-glycans on surface glycoproteins of dendrites shift from immature to mature forms containing sialic acid in response to increased neuronal activation. In exploring the basis of these N-glycosylation alterations, we discovered that they result from the growth and proliferation of Golgi satellites scattered throughout the dendrite. Golgi satellites that formed during neuronal excitation were in close association with endoplasmic reticulum (ER) exit sites and early endosomes and contained glycosylation machinery without the Golgi structural protein, GM130. They functioned as distal glycosylation stations in dendrites, terminally modifying sugars either on newly synthesized glycoproteins passing through the secretory pathway or on surface glycoproteins taken up from the endocytic pathway. These activities led to major changes in the dendritic surface of excited neurons, impacting binding and uptake of lectins, as well as causing functional changes in neurotransmitter receptors such as nicotinic acetylcholine receptors. Neural activity thus boosts the activity of the dendrite’s satellite micro-secretory system by redistributing Golgi enzymes involved in glycan modifications into peripheral Golgi satellites. This remodeling of the neuronal surface has potential significance for synaptic plasticity, addiction, and disease.This work was financially supported by NIH RO1 DA035430, DA044760, and DA043361 (WNG) R01 GM104010 (BSG), T32 GM007183 (FV), and Peter F McManus Foundation (WNG)

Visualizing Protein Sorting in the Golgi Apparatus

In this work I investigated the mechanisms governing protein sorting in the Golgi. The cisternal maturation model of Golgi function postulates that anterograde movement of protein cargoes occurs passively during maturation while resident Golgi proteins are recycled via vesicular transport. Despite suggestive evidence, these predictions had not been tested. Cisternal maturation has been visualized in budding yeast; however, there has not been a tool to enable simultaneous visualization of Golgi cisternae and cargo proteins. Therefore, I first engineered a tool to visualize protein cargoes during Golgi maturation. The tool, Erv29- dependent Secretory Cargo (ESCargo), traps a fluorescent protein in aggregates within the ER. The aggregates are dissolved to release the fluorescent protein for synchronous transport through the secretory pathway. I demonstrated this tool behaves like a bona fide secretory cargo by visualizing its sequential localization with early and then late Golgi cisternae, tracking oligosaccharide modifications, and detecting cargo secreted into the medium. With this tool I verified a key prediction of the cisternal maturation model: secretory cargo remains within the lumen of maturing Golgi cisternae. Unexpectedly, I also discovered that secretory cargo can be recycled within the Golgi in a manner dependent on the AP-1 adaptor complex. Next I dissected how different classes of cargo proteins transit the Golgi. Most models postulate that all cargoes uniformly transit the Golgi to the latest stage of the trans-Golgi network (TGN) where they are sorted into vesicles. To test this hypothesis, I modified ESCargo for transport to the vacuole. I found that vacuolar cargoes exit the Golgi at the beginning of the late Golgi stage, significantly earlier than secretory cargoes. The departure was dependent on the Gga adaptors but not the AP-1 adaptor complex. Thus, the sorting of different cargoes is kinetically segregated during Golgi maturation. ESCargo utilizes evolutionarily conserved machinery and should function in other model organisms. I, along with several collaborators, tested the efficacy of ESCargo in Saccharomyces cerevisiae, mammalian tissue culture, Drosophila melanogaster, and the ciliate Tetrahymena thermophila. In all model organisms tested, ESCargo successfully generated a synchronized cohort of fluorescent secretory cargo - thus permitting comparative studies of secretory cargo trafficking between species. Finally I aimed to clarify the role of the AP-1 adaptor complex in intra-Golgi recycling. Using ESCargo, I found that secretory cargo recycling requires AP-1 and the mannosyltransferase Mnn1. Further, I demonstrated that inhibiting endocytosis in AP-1 mutants causes the mislocalization of resident Golgi enzymes and a severe defect in cisternal maturation. These results suggest that endocytosis acts as a backup recycling pathway for leaked late Golgi proteins in AP-1 mutants. Thus, AP-1 mediates an intra-Golgi recycling pathway that drives cisternal maturation. The majority of the data collected in this work is video microscopy. To appreciate the dynamic nature of protein sorting in the Golgi, it is essential to visualize these processes in real time. The movies associated with each chapter are included as supplementary files online. The first frame and a legend are located at the end of each chapter for reference

A microscopy-based kinetic analysis of yeast vacuolar protein sorting

Saccharomyces cerevisiae is amenable to studying membrane traffic by live-cell fluorescence microscopy. We used this system to explore two aspects of cargo protein traffic through prevacuolar endosome (PVE) compartments to the vacuole. First, at what point during Golgi maturation does a biosynthetic vacuolar cargo depart from the maturing cisternae? To address this question, we modified a regulatable fluorescent secretory cargo by adding a vacuolar targeting signal. Traffic of the vacuolar cargo requires the GGA clathrin adaptors, which arrive during the early-to-late Golgi transition. Accordingly, the vacuolar cargo begins to exit the Golgi near the midpoint of maturation, significantly before exit of a secretory cargo. Second, how are cargoes delivered from PVE compartments to the vacuole? To address this question, we tracked biosynthetic and endocytic cargoes after they had accumulated in PVE compartments. The results suggest that stable PVE compartments repeatedly deliver material to the vacuole by a kiss-and-run mechanism

An improved secretion signal enhances the secretion of model proteins from Pichia pastoris

Background: proteins can be secreted from a host organism with the aid of N-terminal secretion signals. The budding yeast Pichia pastoris (Komagataella sp.) is widely employed to secrete proteins of academic and industrial interest. For this yeast, the most commonly used secretion signal is the N-terminal portion of pre-pro-α-factor from Saccharomyces cerevisiae. However, this secretion signal promotes posttranslational translocation into the endoplasmic reticulum (ER), so proteins that can fold in the cytosol may be inefficiently translocated and thus poorly secreted. In addition, if a protein self-associates, the α-factor pro region can potentially cause aggregation, thereby hampering export from the ER. This study addresses both limitations of the pre-pro-α-factor secretion signal. - Results: we engineered a hybrid secretion signal consisting of the S. cerevisiae Ost1 signal sequence, which promotes cotranslational translocation into the ER, followed by the α-factor pro region. Secretion and intracellular localization were assessed using as a model protein the tetrameric red fluorescent protein E2-Crimson. When paired with the α-factor pro region, the Ost1 signal sequence yielded much more efficient secretion than the α-factor signal sequence. Moreover, an allelic variant of the α-factor pro region reduced aggregation of the E2-Crimson construct in the ER. The resulting improved secretion signal enhanced secretion of E2-Crimson up to 20-fold compared to the levels obtained with the original α-factor secretion signal. Similar findings were obtained with the lipase BTL2, which exhibited 10-fold enhanced secretion with the improved secretion signal. - Conclusions: the improved secretion signal confers dramatic benefits for the secretion of certain proteins from P. pastoris. These benefits are likely to be most evident for proteins that can fold in the cytosol and for oligomeric proteins

Activity-dependent Golgi satellite formation in dendrites reshapes the neuronal surface glycoproteome

Activity-driven changes in the neuronal surface glycoproteome are known to occur with synapse formation, plasticity and related diseases, but their mechanistic basis and significance are unclear. Here, we observed that N-glycans on surface glycoproteins of dendrites shift from immature to mature forms containing sialic acid in response to increased neuronal activation. In exploring the basis of these N-glycosylation alterations, we discovered they result from the growth and proliferation of Golgi satellites scattered throughout the dendrite. Golgi satellites that formed during neuronal excitation were in close association with ER exit sites and early endosomes and contained glycosylation machinery without the Golgi structural protein, GM130. They functioned as distal glycosylation stations in dendrites, terminally modifying sugars either on newly synthesized glycoproteins passing through the secretory pathway, or on surface glycoproteins taken up from the endocytic pathway. These activities led to major changes in the dendritic surface of excited neurons, impacting binding and uptake of lectins, as well as causing functional changes in neurotransmitter receptors such as nicotinic acetylcholine receptors. Neural activity thus boosts the activity of the dendrite’s satellite micro-secretory system by redistributing Golgi enzymes involved in glycan modifications into peripheral Golgi satellites. This remodeling of the neuronal surface has potential significance for synaptic plasticity, addiction and disease

Comprehensive genomic profiling of a rare thyroid follicular dendritic cell sarcoma

We previously reported an extremely rare case of follicular dendritic cell sarcoma (FDCS) presented as a thyroid mass. Given the rarity of this disease, there are no personalized and molecularly targeted treatment options due to the lack of knowledge in the genomic makeup of the tumor. A 44- year-old white woman was diagnosed with an extranodal FDCS in thyroid. The patient underwent a total thyroidectomy, central compartment dissection, parathyroid reimplantation, and adjuvant radiation therapy. Tumor DNA sequencing of 236 genes by FoundationOne panel found truncating mutations in PTEN and missense mutations in RET and TP53. However, patientmatched germline DNA was not sequenced which is critical for identification of true somatic mutations. Furthermore, the FoundationOne panel doesn’t measure genomic rearrangements which have been shown to be abundant in sarcomas and are associated with sarcoma tumorigenesis and progression. In the current study, we carried out comprehensive genomic sequencing of the tumor, adjacent normal tissues, and patient-matched blood, in an effort to understand the genomic makeup of this rare extranodal FDCS and to identify potential therapeutic targets. Eighty-one somatic point mutations were identified in tumor but not in adjacent normal tissues or blood. A clonal truncating mutation in the CLTCL1 gene, which stabilizes the mitotic spindle, was likely a driver mutation of tumorigenesis and could explain the extensive copy number aberrations (CNAs) and genomic rearrangements in the tumor including a chr15/chr17 local chromothripsis resulted in 6 expressed fusion genes. The fusion gene HDGFRP3→SHC4 led to a 200-fold increase in the expression of oncogene SHC4 which is a potential target of the commercial drug Dasatinib. Missense mutations in ATM and splice-site mutation in VEGFR1 were also detected in addition to the TP53 missense mutation reported by FoundationOne