RESUMO
The mitochondria-ER-cortex anchor (MECA) forms a tripartite membrane contact site between mitochondria, the endoplasmic reticulum (ER), and the plasma membrane (PM). The core component of MECA, Num1, interacts with the PM and mitochondria via two distinct lipid-binding domains; however, the molecular mechanism by which Num1 interacts with the ER is unclear. Here, we demonstrate that Num1 contains a FFAT motif in its C-terminus that interacts with the integral ER membrane protein Scs2. While dispensable for Num1's functions in mitochondrial tethering and dynein anchoring, the FFAT motif is required for Num1's role in promoting mitochondrial division. Unexpectedly, we also reveal a novel function of MECA in regulating the distribution of phosphatidylinositol-4-phosphate (PI(4)P). Breaking Num1 association with any of the three membranes it tethers results in an accumulation of PI(4)P on the PM, likely via disrupting Sac1-mediated PI(4)P turnover. This work establishes MECA as an important regulatory hub that spatially organizes mitochondria, ER, and PM to coordinate crucial cellular functions.
Assuntos
Retículo Endoplasmático , Mitocôndrias , Fosfatos de Fosfatidilinositol , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Membrana Celular/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Mitocôndrias/metabolismo , Mitocôndrias/genética , Dinâmica Mitocondrial , Fosfatos de Fosfatidilinositol/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Mitochondrial division is critical for maintenance of mitochondrial morphology and cellular homeostasis. Previous studies have suggested that the mitochondria-ER-cortex anchor (MECA), a tripartite membrane contact site between mitochondria, the ER, and the plasma membrane, is involved in mitochondrial division. However, its role is poorly understood. We developed a system to control MECA formation and depletion, which allowed us to investigate the relationship between MECA-mediated contact sites and mitochondrial division. Num1 is the protein that mediates mitochondria-ER-plasma membrane tethering at MECA sites. Using both rapamycin-inducible dimerization and auxin-inducible degradation components coupled with Num1, we developed systems to temporally control the formation and depletion of the native contact site. Additionally, we designed a regulatable Num1-independant mitochondria-PM tether. We found that mitochondria-PM tethering alone is not sufficient to rescue mitochondrial division and that a specific feature of Num1-mediated tethering is required. This study demonstrates the utility of systems that regulate contact-site formation and depletion in studying the biological functions of membrane contact sites.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Membrana Celular/metabolismo , Proteínas Mitocondriais/metabolismoRESUMO
Positioning organelles at the right place and time is critical for their function and inheritance. In budding yeast, mitochondrial and nuclear positioning require the anchoring of mitochondria and dynein to the cell cortex by clusters of Num1. We have previously shown that mitochondria drive the assembly of cortical Num1 clusters, which then serve as anchoring sites for mitochondria and dynein. When mitochondrial inheritance is inhibited, mitochondrial-driven assembly of Num1 in buds is disrupted and defects in dynein-mediated spindle positioning are observed. Using a structure-function approach to dissect the mechanism of mitochondria-dependent dynein anchoring, we found that the EF hand-like motif (EFLM) of Num1 and its ability to bind calcium are required to bias dynein anchoring on mitochondria-associated Num1 clusters. Consistently, when the EFLM is disrupted, we no longer observe defects in dynein activity following inhibition of mitochondrial inheritance. Thus, the Num1 EFLM functions to bias dynein anchoring and activity in nuclear inheritance subsequent to mitochondrial inheritance. We hypothesize that this hierarchical integration of organelle positioning pathways by the Num1 EFLM contributes to the regulated order of organelle inheritance during the cell cycle.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Motivos EF Hand/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Transporte Biológico , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas do Citoesqueleto/fisiologia , Dineínas/metabolismo , Motivos EF Hand/genética , Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Organelas/fisiologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Fuso Acromático/metabolismoRESUMO
The pathways of membrane traffic within the Golgi apparatus are not fully known. This question was addressed using the yeast Saccharomyces cerevisiae, in which the maturation of individual Golgi cisternae can be visualized. We recently proposed that the AP-1 clathrin adaptor mediates intra-Golgi recycling late in the process of cisternal maturation. Here, we demonstrate that AP-1 cooperates with the Ent5 clathrin adaptor to recycle a set of Golgi transmembrane proteins, including some that were previously thought to pass through endosomes. This recycling can be detected by removing AP-1 and Ent5, thereby diverting the AP-1/Ent5-dependent Golgi proteins into an alternative recycling loop that involves traffic to the plasma membrane followed by endocytosis. Unexpectedly, various AP-1/Ent5-dependent Golgi proteins show either intermediate or late kinetics of residence in maturing cisternae. We infer that the AP-1/Ent5 pair mediates two sequential intra-Golgi recycling pathways that define two classes of Golgi proteins. This insight can explain the polarized distribution of transmembrane proteins in the Golgi.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Endocitose , Complexo de Golgi/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Membrana Celular/metabolismo , Cinética , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Rede trans-Golgi/metabolismoRESUMO
Few membrane contact sites have been defined at the molecular level. By using a high-throughput, microscopy-based screen, Eisenberg-Bord, Zung et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202104100) identify Cnm1 as a novel tethering protein that mediates contact between mitochondria and the nuclear ER in response to phospholipid levels.
Assuntos
Mitocôndrias , Membranas Mitocondriais , Membranas , ProteínasRESUMO
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.
Assuntos
Dendritos/metabolismo , Complexo de Golgi/metabolismo , Glicoproteínas de Membrana/metabolismo , Animais , Autoantígenos/metabolismo , Proliferação de Células , Retículo Endoplasmático/metabolismo , Glicosilação , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Polissacarídeos/metabolismo , Proteoma/metabolismo , Ratos , Receptores Nicotínicos/metabolismoRESUMO
Fluorescence microscopy relies on dyes that absorb and then emit photons. In addition to fluorescence, fluorophores can undergo photochemical processes that decrease quantum yield or result in spectral shifts and irreversible photobleaching. Chemical strategies that suppress these undesirable pathways-thereby increasing the brightness and photostability of fluorophores-are crucial for advancing the frontier of bioimaging. Here, we describe a general method to improve small-molecule fluorophores by incorporating deuterium into the alkylamino auxochromes of rhodamines and other dyes. This strategy increases fluorescence quantum yield, inhibits photochemically induced spectral shifts, and slows irreparable photobleaching, yielding next-generation labels with improved performance in cellular imaging experiments.
RESUMO
Nε-lysine acetylation in the ER is an essential component of the quality control machinery. ER acetylation is ensured by a membrane transporter, AT-1/SLC33A1, which translocates cytosolic acetyl-CoA into the ER lumen, and two acetyltransferases, ATase1 and ATase2, which acetylate nascent polypeptides within the ER lumen. Dysfunctional AT-1, as caused by gene mutation or duplication events, results in severe disease phenotypes. Here, we used two models of AT-1 dysregulation to investigate dynamics of the secretory pathway: AT-1 sTg, a model of systemic AT-1 overexpression, and AT-1S113R/+, a model of AT-1 haploinsufficiency. The animals displayed reorganization of the ER, ERGIC, and Golgi apparatus. In particular, AT-1 sTg animals displayed a marked delay in Golgi-to-plasma membrane protein trafficking, significant alterations in Golgi-based N-glycan modification, and a marked expansion of the lysosomal network. Collectively our results indicate that AT-1 is essential to maintain proper organization and engagement of the secretory pathway.
Assuntos
Acetilcoenzima A/genética , Acetiltransferases/genética , Retículo Endoplasmático/genética , Proteínas de Membrana Transportadoras/genética , Acetilcoenzima A/metabolismo , Acetilação , Autofagia/genética , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica/genética , Complexo de Golgi/genética , Complexo de Golgi/patologia , Haploinsuficiência/genética , Humanos , Lisossomos/genética , Mutação/genética , Processamento de Proteína Pós-Traducional/genética , Transporte Proteico/genética , Via Secretória/genéticaRESUMO
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 fluorescent secretory 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 protein ESCargo (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 with many model organisms.
Assuntos
Modelos Biológicos , Proteínas/metabolismo , Via Secretória , Animais , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Neurônios/metabolismo , Transporte Proteico , Ratos , Saccharomyces cerevisiae/metabolismo , Tetrahymena/metabolismoRESUMO
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.
Assuntos
Endossomos/metabolismo , Complexo de Golgi/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/metabolismo , Microscopia de FluorescênciaRESUMO
COPI vesicles mediate Golgi-to-ER recycling, but COPI vesicle arrival sites at the ER have been poorly defined. We explored this issue using the yeast Pichia pastoris. ER arrival sites (ERAS) can be visualized by labeling COPI vesicle tethers such as Tip20. Our results place ERAS at the periphery of COPII-labeled ER export sites (ERES). The dynamics of ERES and ERAS are indistinguishable, indicating that these structures are tightly coupled. Displacement or degradation of Tip20 does not alter ERES organization, whereas displacement or degradation of either COPII or COPI components disrupts ERAS organization. We infer that Golgi compartments form at ERES and then produce COPI vesicles to generate ERAS. As a result, ERES and ERAS are functionally linked to create bidirectional transport portals at the ER-Golgi interface. COPI vesicles likely become tethered while they bud, thereby promoting efficient retrograde transport. In mammalian cells, the Tip20 homologue RINT1 associates with ERES, indicating possible conservation of the link between ERES and ERAS.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Pichia/citologia , Pichia/metabolismo , Transporte BiológicoRESUMO
Golgi cisternal maturation has been visualized by fluorescence imaging of individual cisternae in the yeast Saccharomyces cerevisiae, but those experiments did not track passage of a secretory cargo. The expectation is that a secretory cargo will be continuously present within maturing cisternae as resident Golgi proteins arrive and depart. We tested this idea using a regulatable fluorescent secretory cargo that forms ER-localized aggregates, which dissociate into tetramers upon addition of a ligand. The solubilized tetramers rapidly exit the ER and then transit through early and late Golgi compartments before being secreted. Early Golgi cisternae form near the ER and become loaded with the secretory cargo. As predicted, cisternae contain the secretory cargo throughout the maturation process. An unexpected finding is that a burst of intra-Golgi recycling delivers additional secretory cargo molecules to cisternae during the early-to-late Golgi transition. This recycling requires the AP-1 adaptor, suggesting that AP-1 can recycle secretory cargo proteins as well as resident Golgi proteins.
Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fator de Transcrição AP-1/metabolismo , Transporte Biológico , Microscopia de Fluorescência , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
Budding yeast is an excellent model organism for studying the dynamics of the Golgi apparatus. To characterize Golgi function, it is important to visualize secretory cargo as it traverses the secretory pathway. We describe a recently developed approach that generates fluorescent protein aggregates in the lumen of the yeast endoplasmic reticulum and allows the fluorescent cargo to be solubilized for transport through the Golgi by addition of a small-molecule ligand. We further describe how to generate a yeast strain expressing the regulatable secretory cargo, and we provide protocols for visualizing the cargo by 4D confocal microscopy and immunoblotting. © 2018 by John Wiley & Sons, Inc.
Assuntos
Complexo de Golgi/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Via Secretória , Transporte Biológico , Retículo Endoplasmático/metabolismo , Immunoblotting/métodos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Saccharomyces cerevisiae/metabolismoRESUMO
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.
Assuntos
Pichia/metabolismo , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes/metabolismo , Sequência de AminoácidosRESUMO
The endomembrane system consists of the secretory and endocytic pathways, which communicate by transport to and from the trans-Golgi network (TGN). In mammalian cells, the endocytic pathway includes early, late, and recycling endosomes. In budding yeast, different types of endosomes have been described, but the organization of the endocytic pathway has remained unclear. We performed a spatial and temporal analysis of yeast endosomal markers and endocytic cargoes. Our results indicate that the yeast TGN also serves as an early and recycling endosome. In addition, as previously described, yeast contains a late or prevacuolar endosome (PVE). Endocytic cargoes localize to the TGN shortly after internalization, and manipulations that perturb export from the TGN can slow the passage of endocytic cargoes to the PVE. Yeast apparently lacks a distinct early endosome. Thus, yeast has a simple endocytic pathway that may reflect the ancestral organization of the endomembrane system.
Assuntos
Membrana Celular/metabolismo , Endocitose/fisiologia , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Saccharomycetales/metabolismo , Rede trans-Golgi/metabolismo , Transporte Biológico , Saccharomycetales/crescimento & desenvolvimentoRESUMO
FK506-binding protein (FKBP) is a monomer that binds to FK506, rapamycin, and related ligands. The F36M substitution, in which Phe36 in the ligand-binding pocket is changed to Met, leads to formation of antiparallel FKBP dimers, which can be dissociated into monomers by ligand binding. This FKBP(M) mutant has been employed in the mammalian secretory pathway to generate aggregates that can be dissolved by ligand addition to create cargo waves. However, when testing this approach in yeast, we found that dissolution of FKBP(M) aggregates was inefficient. An improved reversibly dimerizing FKBP formed aggregates that dissolved more readily. This FKBP(L,V) mutant carries the F36L mutation, which increases the affinity of ligand binding, and the I90V mutation, which accelerates ligand-induced dissociation of the dimers. The FKBP(L,V) mutant expands the utility of reversibly dimerizing FKBP.