Trafficking of the myrosinase‐associated protein GLL23 requires NUC/MVP1/GOLD36/ERMO3 and the p24 protein CYB

Proteins detrimental to endoplasmic reticulum (ER) morphology need to be efficiently exported. Here, we identify two mechanisms that control trafficking of Arabidopsis thalianaGLL23, a 43 kDa GDSL‐like lipase implicated in glucosinolate metabolism through its association with the β‐glucosidase myrosinase. Using immunofluorescence, we identified two mutants that showed aberrant accumulation of GLL23: large perinuclear ER aggregates in the nuclear cage (nuc) mutant; and small compartments contiguous with the peripheral ER in the cytoplasmic bodies (cyb) mutant. Live imaging of fluorescently tagged GLL23 confirmed its presence in the nuc and cyb compartments, but lack of fluorescent signals in the wild‐type plants suggested that GLL23 is normally post‐translationally modified for ER export. NUC encodes the MVP1/GOLD36/ERMO3 myrosinase‐associated protein, previously shown to have vacuolar distribution. CYB is an ER and Golgi‐localized p24 type I membrane protein component of coat protein complex (COP) vesicles, animal and yeast homologues of which are known to be involved in selective cargo sorting for ER–Golgi export. Without NUC, GLL23 accumulates in the ER this situation suggests that NUC is in fact active in the ER. Without CYB, both GLL23 and NUC were found to accumulate in cyb compartments, consistent with a role for NUC in GLL23 processing and indicated that GLL23 is the likely sorting target of the CYB p24 protein.     

A perturbation in glutathione biosynthesis disrupts endoplasmic reticulum morphology and secretory membrane traffic in Arabidopsis thaliana

To identify potentially novel and essential components of plant membrane trafficking mechanisms we performed a GFP-based forward genetic screen for seedling-lethal biosynthetic membrane trafficking mutants in Arabidopsis thaliana. Amongst these mutants, four recessive alleles of GSH2, which encodes glutathione synthase (GSH2), were recovered. Each allele was characterized by loss of the typical polygonal endoplasmic reticulum (ER) network and the accumulation of swollen ER-derived bodies which accumulated a soluble secretory marker. Since GSH2 is responsible for converting γ-glutamylcysteine (γ-EC) to glutathione (GSH) in the glutathione biosynthesis pathway, gsh2 mutants exhibited γ-EC hyperaccumulation and GSH deficiency. Redox-sensitive GFP revealed that gsh2 seedlings maintained redox poise in the cytoplasm but were more sensitive to oxidative challenge. Genetic and pharmacological evidence indicated that γ-EC accumulation rather than GSH deficiency was responsible for the perturbation of ER morphology. Use of soluble and membrane-bound ER markers suggested that the swollen ER bodies were derived from ER fusiform bodies. Despite the gross perturbation of ER morphology, gsh2 seedlings did not suffer from constitutive oxidative ER stress or lack of an unfolded protein response, and homozygotes for the weakest allele could be propagated. The link between glutathione biosynthesis and ER morphology and function is discussed.     

Trafficking of the amino acid transporter B0,+ (SLC6A14) to the plasma membrane involves an exclusive interaction with SEC24C for its exit from the endoplasmic reticulum

A plasma membrane amino acid transporter B^0,+ (ATB^0,+), encoded by the SLC6A14 gene, is specific for neutral and basic amino acids. It is up-regulated in several types of malignant cancers. Neurotransmitter transporters of the SLC6 family interact with specific SEC24 proteins of the COPII complex along their pathway from the endoplasmic reticulum (ER) to Golgi. This study focused on the possible role of SEC24 proteins in ATB^0,+ trafficking. Rat ATB^0,+ was expressed in HEK293 cells, its localization and trafficking were examined by Western blot, deglycosylation, immunofluorescence (co-localization with ER and trans-Golgi markers) and biotinylation. The expression of ATB^0,+ at the plasma membrane was decreased by dominant negative mutants of SAR1, a GTPase, whose activity triggers the formation of the COPII complex. ATB^0,+ co-precipitated with SEC24C (but not with the remaining isoforms A, B and D). This interaction was confirmed by immunocytochemistry and the proximity ligation assay. Co-localization of SEC24C with endogenous ATB^0,+ was also observed in MCF-7 breast cancer cells. Contrary to the endogenous transporter, part of the overexpressed ATB^0,+ is directed to proteolysis, a process significantly reversed by a proteasome inhibitor bortezomib. Co-transfection with a SEC24C dominant negative mutant attenuated ATB^0,+ expression at the plasma membrane, due to proteolytic degradation. These results support a hypothesis that lysine at position +2 downstream of the ER export “RI” motif on the cargo protein is crucial for SEC24C binding and for further trafficking to the Golgi. Moreover, there is an equilibrium between ER export and degradation mechanisms in case of overexpressed transporter.     

Collagen has a unique SEC24 preference for efficient export from the endoplasmic reticulum

SEC24 is mainly involved in cargo sorting during COPII vesicle assembly. There are four SEC24 paralogs (A–D) in vertebrates, which are classified into two subgroups (SEC24A/B and SEC24C/D). Pathological mutations in SEC24D cause osteogenesis imperfecta with craniofacial dysplasia in humans. sec24d mutant fish also recapitulate the phenotypes. Consistent with the skeletal phenotypes, the secretion of collagen was severely defective in mutant fish, emphasizing the importance of SEC24D in collagen secretion. However, SEC24D patient-derived fibroblasts show only a mild secretion phenotype, suggesting tissue-specificity in the secretion process. Using Sec24d KO mice and cultured cells, we show that SEC24A and SEC24B also contribute to endoplasmic reticulum (ER) export of procollagen. In contrast, fibronectin 1 requires either SEC24C or SEC24D for ER export. On the basis of our results, we propose that procollagen interacts with multiple SEC24 paralogs for efficient export from the ER, and that this is the basis for tissue-specific phenotypes resulting from SEC24 paralog deficiency.     

Sfb2p, a yeast protein related to Sec24p, can function as a constituent of COPII coats required for vesicle budding from the endoplasmic reticulum

The COPII coat is required for vesicle budding from the endoplasmic reticulum (ER), and consists of two heterodimeric subcomplexes, Sec23p/Sec24p, Sec13p/Sec31p, and a small GTPase, Sar1p. We characterized a yeast mutant, anu1 (abnormal nuclear morphology) exhibiting proliferated ER as well as abnormal nuclear morphology at the restrictive temperature. Based on the finding that ANU1 is identical to SEC24, we confirmed a temperature-sensitive protein transport from the ER to the Golgi in anu1-1/sec24-20 cells. Overexpression of SFB2, a SEC24 homologue with 56% identity, partially suppressed not only the mutant phenotype of sec24-20 cells but also rescued the SEC24-disrupted cells. Moreover, the yeast two-hybrid assay revealed that Sfb2p, similarly to Sec24p, interacted with Sec23p. In SEC24-disrupted cells rescued by overexpression of SFB2, some cargo proteins were still retained in the ER, while most of the protein transport was restored. Together, these findings strongly suggest that Sfb2p functions as the component of COPII coats in place of Sec24p, and raise the possibility that each member of the SEC24 family of proteins participates directly and/or indirectly in cargo-recognition events with its own cargo specificity at forming ER-derived vesicles.     

Mammalian COPII Coat Component SEC24C Is Required for Embryonic Development in Mice

COPII-coated vesicles mediate the transport of newly synthesized proteins from the endoplasmic reticulum to the Golgi. SEC24 is the COPII component primarily responsible for recruitment of protein cargoes into nascent vesicles. There are four Sec24 paralogs in mammals, with mice deficient in SEC24A, -B, and -D exhibiting a wide range of phenotypes. We now report the characterization of mice with deficiency in the fourth Sec24 paralog, SEC24C. Although mice haploinsufficient for Sec24c exhibit no apparent abnormalities, homozygous deficiency results in embryonic lethality at approximately embryonic day 7. Tissue-specific deletion of Sec24c in hepatocytes, pancreatic cells, smooth muscle cells, and intestinal epithelial cells results in phenotypically normal mice. Thus, SEC24C is required in early mammalian development but is dispensable in a number of tissues, likely as a result of compensation by other Sec24 paralogs. The embryonic lethality resulting from loss of SEC24C occurs considerably later than the lethality previously observed in SEC24D deficiency; it is clearly distinct from the restricted neural tube phenotype of Sec24b null embryos and the mild hypocholesterolemic phenotype of adult Sec24a null mice. Taken together, these results demonstrate that the four Sec24 paralogs have developed unique functions over the course of vertebrate evolution.     

ONTOGENY,HISTOCHEMISTRY AND FINE STRUCTURE OF CELLULAR INCLUSIONS IN VEGETATIVE CELLS OF ANTITHAMNION DEFECTUM (CERAMIACEAE,RHODOPHYTA)1

The ultrastructure and histochemistry of developing and mature cell inclusions in vegetative cells of Antithamnion defectum Kylin were examined. Those studied were chloroplast inclusions, cytoplasmic crystals and spherical bodies within the vacuole. Chloroplasts of mature vegetative cells contain an interthylakoidal, apparently noncrystalline deposit of undetermined chemical identity. The bodies are parallel to the long axis of the plastid, are square (0.13 μm) in cross-section, and up to 3 μm long. Spherical vacuolar bodies (0.5–1.5 μum diam) are formed during early stages of vacuole formation by accumulation of protein deposits in swelling endoplasmic reticulum (ER) cisternae. Swelling of smooth ER contiguous to the ER containing the deposits results in the vacuole enclosing the spherical bodies. In mature cells, vesicles appear to be secreted into the preformed vacuole. Cytoplasmic proteinaceous crystalloids develop without a bounding membrane and may serve as protein reserves.     

PDI reductase acts on Akita mutant proinsulin to initiate retrotranslocation along the Hrd1/Sel1L-p97 axis

In mutant INS gene–induced diabetes of youth (MIDY), characterized by insulin deficiency, MIDY proinsulin mutants misfold and fail to exit the endoplasmic reticulum (ER). Moreover, these mutants bind and block ER exit of wild-type (WT) proinsulin, inhibiting insulin production. The ultimate fate of ER-entrapped MIDY mutants is unclear, but previous studies implicated ER-associated degradation (ERAD), a pathway that retrotranslocates misfolded ER proteins to the cytosol for proteasomal degradation. Here we establish key ERAD machinery components used to triage the Akita proinsulin mutant, including the Hrd1-Sel1L membrane complex, which conducts Akita proinsulin from the ER lumen to the cytosol, and the p97 ATPase, which couples the cytosolic arrival of proinsulin with its proteasomal degradation. Surprisingly, we find that protein disulfide isomerase (PDI), the major protein oxidase of the ER lumen, engages Akita proinsulin in a novel way, reducing proinsulin disulfide bonds and priming the Akita protein for ERAD. Efficient PDI engagement of Akita proinsulin appears linked to the availability of Hrd1, suggesting that retrotranslocation is coordinated on the lumenal side of the ER membrane. We believe that, in principle, this form of diabetes could be alleviated by enhancing the targeting of MIDY mutants for ERAD to restore WT insulin production.     

AN ELECTRON MICROSCOPE STUDY OF THE ENDOPLASMIC RETICULUM IN NEWT NOTOCHORD CELLS AFTER DISTURBANCE WITH ULTRASONIC TREATMENT AND SUBSEQUENT REGENERATION

Ultrasonic treatment of the tails of Triturus alpestris tadpoles, at intensities of 8 to 15 watts/cm², at 1 megacycle/sec., for 5 minutes, disrupted the epidermis and caused pycnosis in individual cells of the muscle and neural tube, but caused no damage to the notochord that could be detected by light microscopy. Electron microscopy showed that this ultrasonic treatment disordered nearly all the endoplasmic reticulum (ER) of the notochord cells into irregularly rounded vesicles, but within 3 hours after treatment some parallel arrays of normal endoplasmic reticulum were seen near, and continuous with, the outer nuclear membrane. In addition, a re-ordering of the previously disordered ER took place throughout the cytoplasm, in some cases. A classification was made of the state of the ER as shown in electron micrographs of material fixed immediately, 3, and 24 hours after treatment. This showed that more than half the total endoplasmic reticulum in notochord cells was normal again by 24 hours after treatment.     

Immunochemical studies on the role of the Golgi complex in protein-body formation in rice seeds

Antibodies raised against purified glutelins and prolamines were employed as probes to study the cellular routes by which these proteins are deposited into protein bodies of rice (Oryza sativa L.) endosperm. Three morphologically distinct protein bodies, large spherical, small spherical, and irregularly-shaped, were observed, in agreement with existing reports. Immunocytochemical studies showed the presence of glutelins in the irregularly-shaped protein bodies while the prolamines were found in both the large and small spherical protein bodies. Both the large and small spherical protein bodies, distinguishable by electron density and gold-labeling patterns, appear to be formed by direct deposition of the newly formed proteins into the lumen of the rough endoplasmic reticulum (ER). In contrast, glutelin protein bodies are formed via the Golgi apparatus. Small electron-lucent vesicles are often found at one side of the Golgi. Electron-dense vesicles, whose contents are labeled by glutelin antibody-gold particles, are commonly observed at the distal side of the Golgi apparatus and fuse to form the irregularly shaped protein bodies in endosperm cells. These observations indicate that the transport of rice glutelins from their site of synthesis, the ER, to the site of deposition, the protein bodies, is mediated by the Golgi apparatus.Abbreviations BSA bovine serum albumin - Da dalton - DAF days after flowering - ER endoplasmic reticulum - GL irregularly shaped - L large spherical - S small spherical (protein bodies) - PBS phosphate-buffered saline - PTA phosphotungstic acid     

Pituitary Adenylate Cyclase-activating Polypeptide Type 1 Receptor (PAC1) Gene Is Suppressed by Transglutaminase 2 Activation

Pituitary adenylate cyclase-activating polypeptide (PACAP) functions as a neuroprotective factor through the PACAP type 1 receptor, PAC1. In a previous work, we demonstrated that nerve growth factor augmented PAC1 gene expression through the activation of Sp1 via the Ras/MAPK pathway. We also observed that PAC1 expression in Neuro2a cells was transiently suppressed during in vitro ischemic conditions, oxygen-glucose deprivation (OGD). Because endoplasmic reticulum (ER) stress is induced by ischemia, we attempted to clarify how ER stress affects the expression of PAC1. Tunicamycin, which induces ER stress, significantly suppressed PAC1 gene expression, and salubrinal, a selective inhibitor of the protein kinase RNA-like endoplasmic reticulum kinase signaling pathway of ER stress, blocked the suppression. In luciferase reporter assay, we found that two Sp1 sites were involved in suppression of PAC1 gene expression due to tunicamycin or OGD. Immunocytochemical staining demonstrated that OGD-induced transglutaminase 2 (TG2) expression was suppressed by salubrinal or cystamine, a TG activity inhibitor. Further, the OGD-induced accumulation of cross-linked Sp1 in nuclei was suppressed by cystamine or salubrinal. Together with cystamine, R283, TG2-specific inhibitor, and siRNA specific for TG2 also ameliorated OGD-induced attenuation of PAC1 gene expression. These results suggest that Sp1 cross-linking might be crucial in negative regulation of PAC1 gene expression due to TG2 in OGD-induced ER stress.     

Disruption of the Sec24d Gene Results in Early Embryonic Lethality in the Mouse

Transport of newly synthesized proteins from the endoplasmic reticulum (ER) to the Golgi is mediated by the coat protein complex COPII. The inner coat of COPII is assembled from heterodimers of SEC23 and SEC24. Though mice with mutations in one of the four Sec24 paralogs, Sec24b, exhibit a neural tube closure defect, deficiency in humans or mice has not yet been described for any of the other Sec24 paralogs. We now report characterization of mice with targeted disruption of Sec24d. Early embryonic lethality is observed in mice completely deficient in SEC24D, while a hypomorphic Sec24d allele permits survival to mid-embryogenesis. Mice haploinsufficient for Sec24d exhibit no phenotypic abnormality. A BAC transgene containing Sec24d rescues the embryonic lethality observed in Sec24d-null mice. These results demonstrate an absolute requirement for SEC24D expression in early mammalian development that is not compensated by the other three Sec24 paralogs. The early embryonic lethality resulting from loss of SEC24D in mice contrasts with the previously reported mild skeletal phenotype of SEC24D deficiency in zebrafish and restricted neural tube phenotype of SEC24B deficiency in mice. Taken together, these observations suggest that the multiple Sec24 paralogs have developed distinct functions over the course of vertebrate evolution.     

Defects in the Secretory Pathway and High Ca2+ Induce Multiple P-bodies

mRNA is sequestered and turned over in cytoplasmic processing bodies (PBs), which are induced by various cellular stresses. Unexpectedly, in Saccharomyces cerevisiae, mutants of the small GTPase Arf1 and various secretory pathway mutants induced a significant increase in PB number, compared with PB induction by starvation or oxidative stress. Exposure of wild-type cells to osmotic stress or high extracellular Ca²⁺ mimicked this increase in PB number. Conversely, intracellular Ca²⁺-depletion strongly reduced PB formation in the secretory mutants. In contrast to PB induction through starvation or osmotic stress, PB formation in secretory mutants and by Ca²⁺ required the PB components Pat1 and Scd6, and calmodulin, indicating that different stressors act through distinct pathways. Consistent with this hypothesis, when stresses were combined, PB number did not correlate with the strength of the translational block, but rather with the type of stress encountered. Interestingly, independent of the stressor, PBs appear as spheres of ∼40–100 nm connected to the endoplasmic reticulum (ER), consistent with the idea that translation and silencing/degradation occur in a spatially coordinated manner at the ER. We propose that PB assembly in response to stress occurs at the ER and depends on intracellular signals that regulate PB number.     

Serum- and Glucocorticoid-Inducible Kinase-1 (SGK-1) Plays a Role in Membrane Trafficking in Caenorhabditis elegans

The mammalian serum- and glucocorticoid-inducible kinase SGK1 regulates the endocytosis of ion channels. Here we report that in C. elegans sgk-1 null mutants, GFP-tagged MIG-14/Wntless, the sorting receptor of Wnt, failed to localize to the basolateral membrane of intestinal cells; instead, it was mis-sorted to lysosomes. This effect can be explained in part by altered sphingolipid levels, because reducing glucosylceramide biosynthesis restored the localization of MIG-14::GFP. Membrane traffic was not perturbed in general, as no obvious morphological defects were detected for early endosomes, the Golgi apparatus, and the endoplasmic reticulum (ER) in sgk-1 null animals. The recycling of MIG-14/Wntless through the Golgi might be partially responsible for the observed phenotype because the subcellular distribution of two plasma membrane cargoes that do not recycle through the trans-Golgi network (TGN) was affected to a lesser degree. Consistently, knockdown of the ArfGEF gbf-1 altered the distribution of SGK-1 at the basolateral membrane of intestinal cells. In addition, we found that sgk-1(RNAi) induced unfolded protein response in the ER, suggesting at least an indirect role of SGK-1 early in the secretory pathway. We propose that SGK-1 function is required for lipid homeostasis and that it acts at different intracellular trafficking steps.     

A lifespan-extending form of autophagy employs the vacuole-vacuole fusion machinery

While autophagy is believed to be beneficial for lifespan extension, it is controversial which forms or aspects of autophagy are the responsible ones. We addressed this by analyzing the lifespan of yeast autophagy mutants under caloric restriction, a longevity manipulation. Surprisingly, we discovered that the majority of proteins involved in macro-autophagy and several forms of micro-autophagy were dispensable for lifespan extension. The only autophagy protein that is critical for lifespan extension was Atg15p, a lipase that is located in the endoplasmic reticulum (ER) and transported to vacuoles for disintegrating membranes of autophagic bodies. We further found that vacuole-vacuole fusion was required for lifespan extension, which was indicated by the shortened lifespan of mutants missing proteins (ypt7Δ, nyv1Δ, vac8Δ) or lipids (erg6Δ) involved in fusion. Since a known function of vacuole-vacuole fusion is the maintenance of the vacuole membrane integrity, we analyzed aged vacuoles and discovered that aged cells had altered vacuolar morphology and accumulated autophagic bodies, suggesting that certain forms of autophagy do contribute to longevity. Like aged cells, erg6Δ accumulated autophagic bodies, which is likely caused by a defect in lipase instead of proteases due to the existence of multiple vacuolar proteases. Since macro-autophagy is not blocked by erg6Δ, we propose that a new form of autophagy transports Atg15p via the fusion of vacuoles with vesicles derived from ER, and we designate this putative form of autophagy as secretophagy. Pending future biochemical studies, the concept of secretophagy may provide a mechanism for autophagy in lifespan extension.     

Nuclear pore complex integrity requires Lnp1, a regulator of cortical endoplasmic reticulum

The nuclear envelope (NE) and endoplasmic reticulum (ER) are components of the same contiguous membrane system and yet have distinct cellular functions. Mounting evidence suggests roles for some ER proteins in the NE for proper nuclear pore complex (NPC) structure and function. In this study, we identify a NE role in Saccharomyces cerevisiae for Lnp1 and Sey1, proteins required for proper cortical ER formation. Both lnp1Δ and sey1Δ mutants exhibit synthetic genetic interactions with mutants in genes encoding key NPC structural components. Both Lnp1 and Sey1 physically associate with other ER components that have established NPC roles, including Rtn1, Yop1, Pom33, and Per33. Of interest, lnp1Δ rtn1Δ mutants but not rtn1Δ sey1Δ mutants exhibit defects in NPC distribution. Furthermore, the essential NPC assembly factor Ndc1 has altered interactions in the absence of Sey1. Lnp1 dimerizes in vitro via its C-terminal zinc finger motif, a property that is required for proper ER structure but not NPC integrity. These findings suggest that Lnp1''s role in NPC integrity is separable from functions in the ER and is linked to Ndc1 and Rtn1 interactions.     

Mechanistic insights in recov-ER-phagy: micro-ER-phagy to recover from stress

ABSTRACT

Physiological and pathological stresses may cause swelling of the endoplasmic reticulum (ER), a biosynthetic organelle in eukaryotic cells. Upon conclusion of the stress, ER size and content return to physiological levels. The translocon component SEC62 decorates the portions of excess ER that must be cleared from cells. Our recent paper highlights the role of endosomal sorting complex required for transport (ESCRT)-III-driven micro-ER-phagy in ER remodeling during cell recovery from ER stress.     

Rice protein-body formation: all types are initiated by dilation of the endoplasmic reticulum

The ultrastructure of protein deposition in the starchy endosperm of developing rice (Oryza sativa L.) grains was examined in conventionally fixed (glutaraldehyde and osmium tetroxide) tissues and also in thick sections (0.3 m) of zinc iodide-osmium tetroxide post-fixed tissue. Three types of previously characterised protein body were observed and it was shown that each type was initiated by dilations of the endoplasmic reticulum. Crystalline type protein bodies were initiated by a ribosome-free dilation from rough cisternal endoplasmic reticulum and developed by inclusion of protein from dictyosome-derived vesicles. The large spherical and small spherical protein bodies developed within the cisternae of the rough endoplasmic reticulum.Abbreviations Cr crystalline protein body - DAF days after fertilization - ER endoplasmic reticulum - Ls large spherical protein body - Ss small spherical protein body - ZIO zinc iodide-osmium tetroxide