Cytochrome c release and endoplasmic reticulum stress are involved in caspase-dependent apoptosis induced by G418

G418 is used extensively in transfection experiments to select eukaryotic cells that have acquired neomycin resistance genes, but the mechanism is still elusive. To investigate this, we treated normal rat kidney cells with G418 for 3 days and found that the cells presented typical apoptotic features such as cell shrinkage, nuclear fragmentation, and caspase-3 activation. However, there was no low-molecular DNA ladder. The pan caspase inhibitor z-VAD-fmk completely inhibited this type of apoptosis, suggesting a caspase-dependent mechanism. Caspase cascades in apoptosis induced by G418 were initiated by at least two pathways: the release of cytochrome c from mitochondria, which was observed under confocal microscopy, and endoplasmic reticulum stress, demonstrated by the increase in Ca²⁺ concentration and the cleavage of m-calpain and procaspase-12. Both pathways activated caspase-9. Inhibition of caspase-9 activity by z-LEHD-fmk prevented most of the cells from apoptosis, and E-64d, an inhibitor of calpain accentuated this block. The cleavage of casapse-9 and caspase-12 was blocked only by simultaneous application of z-VAD-fmk and E-64d, but not by either alone. E-64d did not prevent the release of cytochrome c. These results indicated that these two pathways were independent of each other. Received 1 April 2004; received after revision 21 April 2004; accepted 26 May 2004     

Polypeptide translocation machinery of the yeast endoplasmic reticulum

Proteins enter the secretory pathway by two general routes. In one, the complete polypeptide is made in the cytoplasm and held in an incompletely folded state by chaperoning adenosine triphosphatases (ATPases) such as hsp70. InSaccharomyces cerevisiae, fully synthesized secretory precursors engage the endoplasmic reticulum (ER) membrane by interaction with a set of Sec proteins comprising the polypeptide translocation apparatus (Sec61p, Sec62p, Sec63p, Sec71p, Sec72p). Productive interaction requires displacement of hsp70 from the precursor, a reaction that is facilitated by Ydj1p, a homologue of theEscherichia coli DnaJ protein. Both DnaJ and Ydj1p regulate chaperone activity by stimulating the ATPase activity of their respective hsp70 partners (E. coli DnaK andS. cerevisiae Ssa1p, resepectively). In the ER lumen, another hsp70 chaperone, BiP, binds ATP and interacts with the ER membrane via its contact with a peptide loop of Sec63p. This loop represents yet another DnaJ homologue in that it contains a region of 70 residue similarity to the J box, the most conserved region of the DnaJ family of proteins. In the presence of ATP, under conditions in which BiP can bind to Sec63p, the secretory precursor passes from the cytosol into the lumen through a membrane channel formed by Sec61 p. A second route to the membrane pore that is used by many other secretory precursors, particularly in mammalian cells, requires that the polypeptide engage the ER membrane as the nascent chain emerges from the ribosome. Such cotranslational translocation bypasses the need for certain Sec proteins, instead utilizing an alternate set of cytosolic and membrane factors that allows the nascent chain to be inserted directly into the Sec61p channel.     

A concept of intracellular transmission of excitation by means of the endoplasmic reticulum

Zusammenfassung Die morphologische Beobachtung von getrennten cytoplasmatischen Phasen in geschlossenen Membranen führt zur Annahme von Membranpotentialen innerhalb der Zelle. Schreibt man den Membranen des endoplasmatischen Reticulums die Eigenschaften der Plasmamembran zu, so leiten sie die Erregung ins Zellinnere und dienen als Potentialspeicher.     

Mechanisms for exporting large-sized cargoes from the endoplasmic reticulum

Cargo proteins exported from the endoplasmic reticulum to the Golgi apparatus are typically transported in coat protein complex II (COPII)-coated vesicles of 60–90 nm diameter. Several cargo molecules including collagens and chylomicrons form structures that are too large to be accommodated by these vesicles, but their secretion still requires COPII proteins. Here, we first review recent progress on large cargo secretions derived especially from animal models and human diseases, which indicate the importance of COPII proteins. We then discuss the recent isolation of specialized factors that modulate the process of COPII-dependent cargo formation to facilitate the exit of large-sized cargoes from the endoplasmic reticulum. Based on these findings, we propose a model that describes the importance of the GTPase cycle for secretion of oversized cargoes. Next, we summarize reports that describe the structures of COPII proteins and how these results provide insight into the mechanism of assembly of the large cargo carriers. Finally, we discuss what issues remain to be solved in the future.     

Extensive endoplasmic reticulum: a distinguishing feature of auxin-producing plant cells

Résumé Le cytoplasme des cellules végétales capables de synthétiser l'auxine est caractérisé par l'abondance du réticulum endoplasmique. Les cellules dont la croissance est stimulée par de hautes concentrations de cette hormone contiennent beaucoup moins de réticulum endoplasmique. D'où l'hypothèse que le lieu de synthèse de l'auxine (dans les cellules capables de cette synthèse) est peut-être le réticulum endoplasmique.

---

---

This work was begun at Manhattan College, Bronx, New York, supported by USPHS grant No. CA 06955, the Anna Fuller fund, and the Christine Sonntag Foundation. The work was continued at the Boyce Thompson Institute, Yonkers, New York, and completed at the Wistar Institute with support from USPHS grant No. CA 10815.

---

I am grateful to Dr.G. Hagen for theNicotiana tissues, Dr.J. Lippincott for theP. savastanoi cultures and to Mrs.B. di Gregorio andE. Manasse for competent technical assistance.     

From the endoplasmic reticulum to the plasma membrane: mechanisms of CFTR folding and trafficking

CFTR biogenesis starts with its co-translational insertion into the membrane of endoplasmic reticulum and folding of the cytosolic domains, towards the acquisition of a fully folded compact native structure. Efficiency of this process is assessed by the ER quality control system that allows the exit of folded proteins but targets unfolded/misfolded CFTR to degradation. If allowed to leave the ER, CFTR is modified at the Golgi and reaches the post-Golgi compartments to be delivered to the plasma membrane where it functions as a cAMP- and phosphorylation-regulated chloride/bicarbonate channel. CFTR residence at the membrane is a balance of membrane delivery, endocytosis, and recycling. Several adaptors, motor, and scaffold proteins contribute to the regulation of CFTR stability and are involved in continuously assessing its structure through peripheral quality control systems. Regulation of CFTR biogenesis and traffic (and its dysregulation by mutations, such as the most common F508del) determine its overall activity and thus contribute to the fine modulation of chloride secretion and hydration of epithelial surfaces. This review covers old and recent knowledge on CFTR folding and trafficking from its synthesis to the regulation of its stability at the plasma membrane and highlights how several of these steps can be modulated to promote the rescue of mutant CFTR.     

Signaling pathways between the plasma membrane and endoplasmic reticulum calcium stores

This review discusses multiple ways in which the endoplasmic reticulum participates in and is influenced by signal transduction pathways. The endoplasmic reticulum provides a Ca2+ store that can be mobilized either by calcium-induced calcium release or by the diffusible messenger inositol 1,4,5-trisphosphate. Depletion of endoplasmic reticulum Ca2+ stores provides a signal that activates surface membrane Ca2+ channels, a process known as capacitative calcium entry. Depletion of endoplasmic reticulum stores can also signal long-term cellular responses such as gene expression and programmed cell death or apoptosis. In addition to serving as a source of cellular signals, the endoplasmic reticulum is also functionally and structurally modified by the Ca2+ and protein kinase C pathways. Elevated cytoplasmic Ca2+ causes a rearrangement and fragmentation of endoplasmic reticulum membranes. Protein kinase C activation reduces the storage capacity of the endoplasmic reticulum Ca2+ pool. In some cell types, protein kinase C inhibits capacitative calcium entry. Protein kinase C activation also protects the endoplasmic reticulum from the structural effects of high cytoplasmic Ca2+. The emerging view is one of a complex network of pathways through which the endoplasmic reticulum and the Ca2+ and protein kinase C signaling pathways interact at various levels regulating cellular structure and function.     

Similar paracrystals of endoplasmic reticulum in the photoemitters and the photoreceptors of scale-worms

Summary Paracrystals of tubular endoplasmic reticulum are the characteristic of the luminous cells in the elytra of polynoïd worms, and they are the sources of luminescence as well as of fluorescence. Structurally similar paracrystals are now found to be the main constituent of the lens in the eyes of all the Aphroditidae.     

Chaperone-mediated autophagy: machinery,regulation and biological consequences

Degradation of dysfunctional intracellular components in the lysosome system can occur through three different pathways, i.e., macroautophagy, microautophagy and chaperone-mediated autophagy (CMA). In this review, we focus on CMA, a type of autophagy distinct from the other two autophagic pathways owing to its selectivity, saturability and competitivity by which a subset of long-lived cytosolic soluble proteins are directly delivered into the lysosomal lumen via specific receptors. CMA participates in quality control to maintain normal cell functions by clearing “old” proteins and provides energy to cells under nutritional stress. Deregulation of CMA has recently been shown to underlie some diseases, especially neurodegenerative disorders for which the decline with age in the activity of CMA may become a major aggravating factor. Therefore, targeting aberrant alteration in CMA under pathological conditions could serve as a potential therapeutic strategy for treating related diseases.     

Effect of praseodymium on drug metabolism in rat liver smooth and rough endoplasmic reticulum

Summary A small i.v. dose (3 mg/kg) of a light lanthanon, praseodymium, impairs the drug metabolizing capacity of both the smooth and rough fractions of rat liver endoplasmic reticulum. This decrease in the activity of drug metabolizing enzymes and in the amount of cytochromes P-450 and b₅ is more pronounced in the rough endoplasmic reticulum fraction.     

Role of the nuclear membrane in smooth endoplasmic reticulum formation in white rat pinealocytes

Résumé La glande pinéale du rat blanc est étudiée ici au microscope électronique. L'association observée entre les vésicules agranulaires et les «blebs» de la membrane nucléaire est un argument en faveur de la théorie selon laquelle l'origine du reticulum endoplasmique est à rechercher dans la membrane nucléaire.     

APP dimer formation is initiated in the endoplasmic reticulum and differs between APP isoforms

The amyloid precursor protein (APP) is part of a larger gene family, which has been found to form homo- or heterotypic complexes with its homologues, whereby the exact molecular mechanism and origin of dimer formation remains elusive. In order to assess the cellular location of dimerization, we have generated a cell culture model system in CHO-K1 cells, stably expressing human APP, harboring dilysine-based organelle sorting motifs [KKAA-endoplasmic reticulum (ER); KKFF-Golgi], accomplishing retention within early secretory compartments. We show that APP exists as disulfide-bonded dimers upon ER retention after it was isolated from cells, and analyzed by SDS-polyacrylamide gel electrophoresis under non-reducing conditions. In contrast, strong denaturing and reducing conditions, or deletion of the E1 domain, resulted in the disappearance of those dimers. Thus we provide first evidence that a fraction of APP can associate via intermolecular disulfide bonds, likely generated between cysteines located in the extracellular E1 domain. We particularly visualize APP dimerization itself and identified the ER as subcellular compartment of its origin using biochemical or split GFP approaches. Interestingly, we also found that minor amounts of SDS-resistant APP dimers were located to the cell surface, revealing that once generated in the oxidative environment of the ER, dimers remained stably associated during transport. In addition, we show that APP isoforms encompassing the Kunitz-type protease inhibitor (KPI) domain exhibit a strongly reduced ability to form cis-directed dimers in the ER, whereas trans-mediated cell aggregation of Drosophila Schneider S2-cells was isoform independent. Thus, suggesting that steric properties of KPI-APP might be the cause for weaker cis-interaction in the ER, compared to APP695. Finally, we provide evidence that APP/APLP1 heterointeractions are likewise initiated in the ER.     

BiP (GRP78), an essential hsp70 resident protein in the endoplasmic reticulum

BiP is a constitutively-expressed resident protein of the endoplasmic, reticulum (ER) of all eucaryotic cells, and belongs to the highly conserved hsp70 protein family. In the ER, BiP is involved in polypeptide translocation, protein folding and presumably protein degradation as well. These functions are essential to cell viability, as has been shown for yeast. In this review, I will summarize the structural features of hsp70 proteins and focus on those experiments which revealed the biological function of BiP.     

Glycoprotein misfolding in the endoplasmic reticulum: identification of released oligosaccharides reveals a second ER-associated degradation pathway for Golgi-retrieved proteins

Endoplasmic reticulum-associated degradation (ERAD) is a key cellular process whereby misfolded proteins are removed from the endoplasmic reticulum (ER) for subsequent degradation by the ubiquitin/proteasome system. In the present work, analysis of the released, free oligosaccharides (FOS) derived from all glycoproteins undergoing ERAD, has allowed a global estimation of the mechanisms of this pathway rather than following model proteins through degradative routes. Examining the FOS produced in endomannosidase-compromised cells following α-glucosidase inhibition has revealed a mechanism for clearing Golgi-retrieved glycoproteins that have failed to enter the ER quality control cycle. The Glc₃Man₇GlcNAc₂ FOS species has been shown to be produced in the ER lumen by a mechanism involving a peptide: N-glycanase-like activity, and its production was sensitive to disruption of Golgi-ER trafficking. The detection of this oligosaccharide was unaffected by the overexpression of EDEM1 or cytosolic mannosidase, both of which increased the production of previously characterised cytosolically localised FOS. The lumenal FOS identified are therefore distinct in their production and regulation compared to FOS produced by the conventional route of misfolded glycoproteins directly removed from the ER. The production of such lumenal FOS is indicative of a novel degradative route for cellular glycoproteins that may exist under certain conditions.