215 research outputs found
The Mechanisms of Vesicle Budding and Fusion
AbstractGenetic and biochemical analyses of the secretory pathway have produced a detailed picture of the molecular mechanisms involved in selective cargo transport between organelles. This transport occurs by means of vesicular intermediates that bud from a donor compartment and fuse with an acceptor compartment. Vesicle budding and cargo selection are mediated by protein coats, while vesicle targeting and fusion depend on a machinery that includes the SNARE proteins. Precise regulation of these two aspects of vesicular transport ensures efficient cargo transfer while preserving organelle identity
Noncytotoxic orange and red/green derivatives of DsRed-Express2 for whole-cell labeling
<p>Abstract</p> <p>Background</p> <p>Whole-cell labeling is a common application of fluorescent proteins (FPs), but many red and orange FPs exhibit cytotoxicity that limits their use as whole-cell labels. Recently, a tetrameric red FP called DsRed-Express2 was engineered for enhanced solubility and was shown to be noncytotoxic in bacterial and mammalian cells. Our goal was to create derivatives of this protein with different spectral properties.</p> <p>Results</p> <p>Building on previous studies of DsRed mutants, we created two DsRed-Express2 derivatives: E2-Orange, an orange FP, and E2-Red/Green, a dual-color FP with both red and green emission. We show that these new FPs retain the low cytotoxicity of DsRed-Express2. In addition, we show that these new FPs are useful as second or third colors for flow cytometry and fluorescence microscopy.</p> <p>Conclusion</p> <p>E2-Orange and E2-Red/Green will facilitate the production of healthy, stably fluorescent cell lines and transgenic organisms for multi-color labeling studies.</p
A Role for Actin, Cdc1p, and Myo2p in the Inheritance of Late Golgi Elements in \u3cem\u3eSaccharomyces cerevisiae\u3c/em\u3e
In Saccharomyces cerevisiae, Golgi elements are present in the bud very early in the cell cycle. We have analyzed this Golgi inheritance process using fluorescence microscopy and genetics. In rapidly growing cells, late Golgi elements show an actin-dependent concentration at sites of polarized growth. Late Golgi elements are apparently transported into the bud along actin cables and are also retained in the bud by a mechanism that may involve actin. A visual screen for mutants defective in the inheritance of late Golgi elements yielded multiple alleles of CDC1. Mutations in CDC1 severely depolarize the actin cytoskeleton, and these mutations prevent late Golgi elements from being retained in the bud. The efficient localization of late Golgi elements to the bud requires the type V myosin Myo2p, further suggesting that actin plays a role in Golgi inheritance. Surprisingly, early and late Golgi elements are inherited by different pathways, with early Golgi elements localizing to the bud in a Cdc1p- and Myo2p-independent manner. We propose that early Golgi elements arise from ER membranes that are present in the bud. These two pathways of Golgi inheritance in S. cerevisiae resemble Golgi inheritance pathways in vertebrate cells
Golgi Inheritance in Small Buds of Saccharomyces Cerevisiae Is Linked to Endoplasmic Reticulum Inheritance
According to the cisternal maturation hypothesis, endoplasmic reticulum (ER)-derived membranes nucleate new Golgi cisternae. The yeast Saccharomyces cerevisiae offers a unique opportunity to test this idea because small buds contain both ER and Golgi structures early in the cell cycle. We previously predicted that mutants defective in ER inheritance also would show defects in Golgi inheritance. Surprisingly, studies of S. cerevisiae have not revealed the expected link between ER and Golgi inheritance. Here, we revisit this issue by generating mutant strains in which many of the small buds are devoid of detectable ER. These strains also show defects in the inheritance of both early and late Golgi cisternae. Strikingly, virtually all of the buds that lack ER also lack early Golgi cisternae. Our results fit with the idea that membranes exported from the ER coalesce with vesicles derived from existing Golgi
compartments to generate new Golgi cisternae. This basic mech- anism of Golgi inheritance may be conserved from yeast to verte- brate cells
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
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
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