scholarly journals Arabidopsis Lunapark proteins are involved in ER cisternae formation

2018 ◽  
Author(s):  
Verena Kriechbaumer ◽  
Emily Breeze ◽  
Charlotte Pain ◽  
Frances Tolmie ◽  
Lorenzo Frigerio ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Confocal Microscopy ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Yeast Cells ◽  
Subcellular Localisation ◽  
Mutant Analysis ◽  
Protein Protein Interaction ◽  
Dynamic Morphology ◽  
Network Organisation

SummaryThe plant endoplasmic reticulum (ER) is crucial to the maintenance of cellular homeostasis. The ER consists of a dynamic and continuously remodelling network of tubules and cisternae. Several conserved membrane proteins have been implicated in formation and maintenance of the ER network in plants, such as RHD3 and the reticulon family of proteins.Despite the recent work in mammalian and yeast cells, the detailed molecular mechanisms of ER network organisation in plants still remain largely unknown. Recently novel ER network-shaping proteins called Lunapark have been identified in yeast and mammalian cells.Here we identify two arabidopsis LNP homologues and investigate their subcellular localisation via confocal microscopy and potential function in shaping the ER network using protein-protein interaction assays and mutant analysis.We show that AtLNP1 overexpression in tobacco leaf epidermal cells mainly labels the three-way junctions (trivia) of the ER network whereas AtLNP2 labels the whole ER. Overexpression of LNP proteins results in an increased abundance of ER cisternae and an lnp1lnp2 amiRNA line displays a less structured ER network.Thus, we hypothesize that AtLNP1 and AtLNP2 are involved in determining the dynamic morphology of the plant ER, possibly by regulating the formation of ER cisternae.

scholarly journals Characterization of constitutive ER-phagy of excess membrane proteins

PLoS Genetics ◽  
2020 ◽  
Vol 16 (12) ◽  
pp. e1009255
Author(s):  
Zhanna Lipatova ◽  
Valeriya Gyurkovska ◽  
Sarah F. Zhao ◽  
Nava Segev
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Human Health ◽  
Mammalian Cells ◽  
Normal Growth ◽  
Integral Membrane Proteins ◽  
Yeast Cells ◽  
Cellular Proteins

Thirty percent of all cellular proteins are inserted into the endoplasmic reticulum (ER), which spans throughout the cytoplasm. Two well-established stress-induced pathways ensure quality control (QC) at the ER: ER-phagy and ER-associated degradation (ERAD), which shuttle cargo for degradation to the lysosome and proteasome, respectively. In contrast, not much is known about constitutive ER-phagy. We have previously reported that excess of integral-membrane proteins is delivered from the ER to the lysosome via autophagy during normal growth of yeast cells. Whereas endogenously expressed ER resident proteins serve as cargos at a basal level, this level can be induced by overexpression of membrane proteins that are not ER residents. Here, we characterize this pathway as constitutive ER-phagy. Constitutive and stress-induced ER-phagy share the basic macro-autophagy machinery including the conserved Atgs and Ypt1 GTPase. However, induction of stress-induced autophagy is not needed for constitutive ER-phagy to occur. Moreover, the selective receptors needed for starvation-induced ER-phagy, Atg39 and Atg40, are not required for constitutive ER-phagy and neither these receptors nor their cargos are delivered through it to the vacuole. As for ERAD, while constitutive ER-phagy recognizes cargo different from that recognized by ERAD, these two ER-QC pathways can partially substitute for each other. Because accumulation of membrane proteins is associated with disease, and constitutive ER-phagy players are conserved from yeast to mammalian cells, this process could be critical for human health.

Visualizing the intracellular membrane system of yeast cells using a laser scanning confocal microscope

1993 ◽  
Vol 51 ◽  
pp. 274-275
Author(s):  
Karen L. McCoy ◽  
Andrew G. Dillin ◽  
Ardythe A. McCracken
Keyword(s):  
Endoplasmic Reticulum ◽  
Yeast Cell ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Intracellular Localization ◽  
Fluorescent Microscopy ◽  
Confocal Microscope ◽  
Yeast Cells ◽  
Intracellular Membrane ◽  
Laser Scanning Confocal Microscope

Progress has been made in developing new preparative techniques for electron microscopic visualization of the intracellular structures of yeast. In addition, development of the laser scanning confocal microscope (LSCM) has provided improved resolution for fluorescent microscopy. We asked whether the LSCM in combination with new preparative techniques could be used for comparable investigative research of the intracellular organizaton of the yeast cell.To investigate this possibility, a BioRad MRC600 LSCM equipped with a krypton/argon laser and integrated computer imaging capabilities, was used to study various dipliod strains of the yeast Saccharomyces cerevisiae. Cells were treated with the lipophilic, cationic fluorescent dye DiOC6 (3,3’-dihexyloxacarbocyanine iodide), which has been used to visualize intracellular membrane structures, and in particular the endoplasmic reticulum of mammalian cells and living yeast cells. Since one of our interests is the intracellular localization of proteins in the yeast cell, we utilized transformed yeast cells expressing a human gene encoding a protein that inappropriately accumulates in the endoplasmic reticulum (ER).

The promoter region of the yeast KAR2 (BiP) gene contains a regulatory domain that responds to the presence of unfolded proteins in the endoplasmic reticulum

1993 ◽  
Vol 13 (2) ◽  
pp. 877-890 ◽  
Author(s):  
K Kohno ◽  
K Normington ◽  
J Sambrook ◽  
M J Gething ◽  
K Mori
Keyword(s):  
Endoplasmic Reticulum ◽  
Heat Shock ◽  
Mammalian Cells ◽  
Yeast Cells ◽  
Regulatory Sequences ◽  
Upstream Sequence ◽  
Unfolded Proteins ◽  
Heat Shock Element ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cis Acting

The endoplasmic reticulum (ER) of eukaryotic cells contains an abundant 78,000-Da protein (BiP) that is involved in the translocation, folding, and assembly of secretory and transmembrane proteins. In the yeast Saccharomyces cerevisiae, as in mammalian cells, BiP mRNA is synthesized at a high basal rate and is further induced by the presence of increased amounts of unfolded proteins in the ER. However, unlike mammalian BiP, yeast BiP is also induced severalfold by heat shock, albeit in a transient fashion. To identify the regulatory sequences that respond to these stimuli in the yeast KAR2 gene that encodes BiP, we have cloned a 1.3-kb segment of DNA from the region upstream of the sequences coding for BiP and fused it to a reporter gene, the Escherichia coli beta-galactosidase gene. Analysis of a series of progressive 5' truncations as well as internal deletions of the upstream sequence showed that the information required for accurate transcriptional regulation of the KAR2 gene in S. cerevisiae is contained within a approximately 230-bp XhoI-DraI fragment (nucleotides -245 to -9) and that this fragment contains at least two cis-acting elements, one (heat shock element [HSE]) responding to heat shock and the other (unfolded protein response element [UPR]) responding to the presence of unfolded proteins in the ER. The HSE and UPR elements are functionally independent of each other but work additively for maximum induction of the yeast KAR2 gene. Lying between these two elements is a GC-rich region that is similar in sequence to the consensus element for binding of the mammalian transcription factor Sp1 and that is involved in the basal expression of the KAR2 gene. Finally, we provide evidence suggesting that yeast cells monitor the concentration of free BiP in the ER and adjust the level of transcription of the KAR2 gene accordingly; this effect is mediated via the UPR element in the KAR2 promoter.

scholarly journals Characterization of Constitutive macro-ER-phagy

2020 ◽  
Author(s):  
Zhanna Lipatova ◽  
Valeriya Gyurkovska ◽  
Sarah F. Zhao ◽  
Nava Segev
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Er Stress ◽  
Mammalian Cells ◽  
Normal Growth ◽  
Integral Membrane Proteins ◽  
Yeast Cells ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

AbstractThirty percent of all cellular proteins are inserted into the endoplasmic reticulum (ER), which spans throughout the cytoplasm. Two well-established stress-induced pathways ensure quality control (QC) at the ER: ER-phagy and ER-associated degradation (ERAD), which shuttle cargo for degradation to the lysosome and proteasome, respectively. In contrast, not much is known about constitutive ER-phagy. We have previously reported that excess of integral-membrane proteins is delivered from the ER to the lysosome via autophagy during normal growth of yeast cells. Here, we characterize this pathway as constitutive ER-phagy. Constitutive and stress-induced ER-phagy share the basic macro-autophagy machinery including the conserved Atgs and Ypt1 GTPase. However, induction of stress-induced autophagy is not needed for constitutive ER-phagy to occur. Moreover, the selective receptors needed for starvation-induced ER-phagy, Atg39 and Atg40, are not required for constitutive ER-phagy and neither these receptors nor their cargos are delivered through it to the vacuole. As for ERAD, while constitutive ER-phagy recognizes cargo different from that recognized by ERAD, these two ER-QC pathways can partially substitute for each other. Because accumulation of membrane proteins is associated with disease, and constitutive ER-phagy players are conserved from yeast to mammalian cells, this process could be critical for human health.Author SummaryAccumulation of excess proteins can lead to their aggregation, which in turn can cause multiple disorders, notably neurodegenerative disease. Nutritional and endoplasmic-reticulum (ER) stress stimulate autophagy and ER-associated degradation (ERAD) to clear excess and misfolded proteins, respectively. However, not much is known about clearance of excess proteins during normal growth. We have previously shown that excess integral-membrane proteins are cleared from the ER by macro-autophagy during normal growth of yeast cells. Here we characterize this pathway as constitutive ER-phagy. While this pathway shares machinery of core Atgs and autophagosomes with nutritional stress-induced ER-phagy, it differs from the latter: It is independent of the stress response and of receptors needed for stress-induced ER-phagy, and while stress-induced ER-phagy is not discriminatory, constitutive ER-phagy has specific cargos. However, when constitutive ER-phagy fails, machinery specific to stress-induced ER-phagy can process its cargo. Moreover, constitutive ER-phagy is also not dependent on ER-stress or the unfolded protein response (UPR) stimulated by this stress, although its failure elicits UPR. Finally, constitutive ER-phagy and ERAD can partially process each other’s cargo upon failure. In summary, constitutive ER-phagy, which clears excess integral-membrane proteins from the ER during normal growth does not require nutritional or ER stress for its function.

scholarly journals Yeast Cells Exposed to Exogenous Palmitoleic Acid Either Adapt to Stress and Survive or Commit to Regulated Liponecrosis and Die

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Karamat Mohammad ◽  
Paméla Dakik ◽  
Younes Medkour ◽  
Mélissa McAuley ◽  
Darya Mitrofanova ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Death ◽  
Lipid Metabolism ◽  
Lipid Droplets ◽  
Palmitoleic Acid ◽  
Molecular Mechanisms ◽  
Neutral Lipids ◽  
Yeast Cells ◽  
Cellular Processes ◽  
Regulated Cell Death

A disturbed homeostasis of cellular lipids and the resulting lipotoxicity are considered to be key contributors to many human pathologies, including obesity, metabolic syndrome, type 2 diabetes, cardiovascular diseases, and cancer. The yeast Saccharomyces cerevisiae has been successfully used for uncovering molecular mechanisms through which impaired lipid metabolism causes lipotoxicity and elicits different forms of regulated cell death. Here, we discuss mechanisms of the “liponecrotic” mode of regulated cell death in S. cerevisiae. This mode of regulated cell death can be initiated in response to a brief treatment of yeast with exogenous palmitoleic acid. Such treatment prompts the incorporation of exogenously added palmitoleic acid into phospholipids and neutral lipids. This orchestrates a global remodeling of lipid metabolism and transfer in the endoplasmic reticulum, mitochondria, lipid droplets, and the plasma membrane. Certain features of such remodeling play essential roles either in committing yeast to liponecrosis or in executing this mode of regulated cell death. We also outline four processes through which yeast cells actively resist liponecrosis by adapting to the cellular stress imposed by palmitoleic acid and maintaining viability. These prosurvival cellular processes are confined in the endoplasmic reticulum, lipid droplets, peroxisomes, autophagosomes, vacuoles, and the cytosol.

Cell Size Control in Plants

2019 ◽  
Vol 53 (1) ◽  
pp. 45-65 ◽  
Author(s):  
Marco D'Ario ◽  
Robert Sablowski
Keyword(s):  
Cell Size ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Growth Control ◽  
Size Control ◽  
Yeast Cells ◽  
Shoot Meristem ◽  
Functional Consequences ◽  
Cell Size Control ◽  
Size Heterogeneity

The genetic control of the characteristic cell sizes of different species and tissues is a long-standing enigma. Plants are convenient for studying this question in a multicellular context, as their cells do not move and are easily tracked and measured from organ initiation in the meristems to subsequent morphogenesis and differentiation. In this article, we discuss cell size control in plants compared with other organisms. As seen from yeast cells to mammalian cells, size homeostasis is maintained cell autonomously in the shoot meristem. In developing organs, vacuolization contributes to cell size heterogeneity and may resolve conflicts between growth control at the cellular and organ levels. Molecular mechanisms for cell size control have implications for how cell size responds to changes in ploidy, which are particularly important in plant development and evolution. We also discuss comparatively the functional consequences of cell size and their potential repercussions at higher scales, including genome evolution.

scholarly journals Reconstituting the Mammalian Apoptotic Switch in Yeast

Genes ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 145 ◽  
Author(s):  
Peter Polčic ◽  
Marek Mentel
Keyword(s):  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Model System ◽  
Yeast Cells ◽  
Mitochondrial Outer Membrane ◽  
Mitochondrial Membranes ◽  
Antiapoptotic Proteins ◽  
The Family ◽  
Induction Of Apoptosis ◽  
Apoptotic Signal

Proteins of the Bcl-2 family regulate the permeabilization of the mitochondrial outer membrane that represents a crucial irreversible step in the process of induction of apoptosis in mammalian cells. The family consists of both proapoptotic proteins that facilitate the membrane permeabilization and antiapoptotic proteins that prevent it in the absence of an apoptotic signal. The molecular mechanisms, by which these proteins interact with each other and with the mitochondrial membranes, however, remain under dispute. Although yeast do not have apparent homologues of these apoptotic regulators, yeast cells expressing mammalian members of the Bcl-2 family have proved to be a valuable model system, in which action of these proteins can be effectively studied. This review focuses on modeling the activity of proapoptotic as well as antiapoptotic proteins of the Bcl-2 family in yeast.

scholarly journals The TIR-domain containing effectors BtpA and BtpB from Brucella abortus block energy metabolism

2019 ◽  
Author(s):  
Julia María Coronas-Serna ◽  
Arthur Louche ◽  
María Rodríguez-Escudero ◽  
Morgane Roussin ◽  
Paul R.C. Imbert ◽  
...  
Keyword(s):  
Energy Metabolism ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Cell Model ◽  
Interleukin 1 ◽  
Effector Proteins ◽  
Host Cells ◽  
Yeast Cells ◽  
Tir Domain ◽  
Tir Domains

ABSTRACTBrucella species are facultative intracellular Gram-negative bacteria relevant to animal and human health. Their ability to establish an intracellular niche and subvert host cell pathways to their advantage depends on the delivery of bacterial effector proteins through a type IV secretion system. Brucella Toll/Interleukin-1 Receptor (TIR)-domain-containing proteins BtpA (also known as TcpB) and BtpB are among such effectors. Although divergent in primary sequence, they interfere with Toll-like receptor (TLR) signaling to inhibit the innate immune responses. However, the molecular mechanisms implicated still remain unclear. To gain insight into the functions of BtpA and BtpB, we expressed them in the budding yeast Saccharomyces cerevisiae as a eukaryotic cell model. We found that both effectors were cytotoxic and that their respective TIR domains were necessary and sufficient for yeast growth inhibition. Growth arrest was concomitant with actin depolymerization, endocytic block and a general decrease in kinase activity in the cell, suggesting a failure in energetic metabolism. Indeed, levels of ATP and NAD+ were low in yeast cells expressing BtpA and BtpB TIR domains, consistent with the recently described enzymatic activity of some TIR domains as NAD+ hydrolases. In human epithelial cells, both BtpA and BtpB expression reduced intracellular total NAD levels. In infected cells, both BtpA and BtpB contributed to reduction of total NAD, indicating that their NAD+ hydrolase functions are active intracellularly during infection. Overall, combining the yeast model together with mammalian cells and infection studies our results show that BtpA and BtpB modulate energy metabolism in host cells through NAD+ hydrolysis, assigning a novel role for these TIR domain-containing effectors in Brucella pathogenesis.

scholarly journals The Saccharomyces cerevisiae SCS2 Gene Product, a Homolog of a Synaptobrevin-Associated Protein, Is an Integral Membrane Protein of the Endoplasmic Reticulum and Is Required for Inositol Metabolism

1998 ◽  
Vol 180 (7) ◽  
pp. 1700-1708 ◽  
Author(s):  
Satoshi Kagiwada ◽  
Kohei Hosaka ◽  
Masayuki Murata ◽  
Jun-ichi Nikawa ◽  
Akira Takatsuki
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Amino Acid ◽  
Gene Product ◽  
Mammalian Cells ◽  
Aplysia Californica ◽  
Integral Membrane Protein ◽  
Yeast Cells ◽  
Eukaryotic Cells ◽  
Genetic Studies

ABSTRACT The Saccharomyces cerevisiae SCS2 gene has been cloned as a suppressor of inositol auxotrophy of CSE1 andhac1/ire15 mutants (J. Nikawa, A. Murakami, E. Esumi, and K. Hosaka, J. Biochem. 118:39–45, 1995) and has homology with a synaptobrevin/VAMP-associated protein, VAP-33, cloned fromAplysia californica (P. A. Skehel, K. C. Martin, E. R. Kandel, and D. Bartsch, Science 269:1580–1583, 1995). In this study we have characterized an SCS2 gene product (Scs2p). The product has a molecular mass of 35 kDa and is C-terminally anchored to the endoplasmic reticulum, with the bulk of the protein located in the cytosol. The disruption of the SCS2 gene causes yeast cells to exhibit inositol auxotrophy at temperatures of above 34°C. Genetic studies reveal that the overexpression of theINO1 gene rescues the inositol auxotrophy of theSCS2 disruption strain. The significant primary structural feature of Scs2p is that the protein contains the 16-amino-acid sequence conserved in yeast and mammalian cells. The sequence is required for normal Scs2p function, because a mutant Scs2p that lacks the sequence does not complement the inositol auxotrophy of theSCS2 disruption strain. Therefore, the Scs2p function might be conserved among eukaryotic cells.

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