GMAP-210, A cis-Golgi network-associated protein, is a minus end microtubule-binding protein

We report that a peripheral Golgi protein with a molecular mass of 210 kD localized at the cis-Golgi network (Rios, R.M., A.M. Tassin, C. Celati, C. Antony, M.C. Boissier, J.C. Homberg, and M. Bornens. 1994. J. Cell Biol. 125:997-1013) is a microtubule-binding protein that associates in situ with a subpopulation of stable microtubules. Interaction of this protein, now called GMAP-210, for Golgi microtubule-associated protein 210, with microtubules in vitro is direct, tight and nucleotide-independent. Biochemical analysis further suggests that GMAP-210 specifically binds to microtubule ends. The full-length cDNA encoding GMAP-210 predicts a protein of 1, 979 amino acids with a very long central coiled-coil domain. Deletion analyses in vitro show that the COOH terminus of GMAP-210 binds to microtubules whereas the NH2 terminus binds to Golgi membranes. Overexpression of GMAP-210-encoding cDNA induced a dramatic enlargement of the Golgi apparatus and perturbations in the microtubule network. These effects did not occur when a mutant lacking the COOH-terminal domain was expressed. When transfected in fusion with the green fluorescent protein, the NH2-terminal domain associated with the cis-Golgi network whereas the COOH-terminal microtubule-binding domain localized at the centrosome. Altogether these data support the view that GMAP-210 serves to link the cis-Golgi network to the minus ends of centrosome-nucleated microtubules. In addition, this interaction appears essential for ensuring the proper morphology and size of the Golgi apparatus.     

Characterization of a cis-Golgi matrix protein, GM130

Antisera raised to a detergent- and salt-resistant matrix fraction from rat liver Golgi stacks were used to screen an expression library from rat liver cDNA. A full-length clone was obtained encoding a protein of 130 kD (termed GM130), the COOH-terminal domain of which was highly homologous to a Golgi human auto-antigen, golgin-95 (Fritzler et al., 1993). Biochemical data showed that GM130 is a peripheral cytoplasmic protein that is tightly bound to Golgi membranes and part of a larger oligomeric complex. Predictions from the protein sequence suggest that GM130 is an extended rod-like protein with coiled-coil domains. Immunofluorescence microscopy showed partial overlap with medial- and trans-Golgi markers but almost complete overlap with the cis-Golgi network (CGN) marker, syntaxin5. Immunoelectron microscopy confirmed this location showing that most of the GM130 was located in the CGN and in one or two cisternae on the cis-side of the Golgi stack. GM130 was not re-distributed to the ER in the presence of brefeldin A but maintained its overlap with syntaxin5 and a partial overlap with the ER- Golgi intermediate compartment marker, p53. Together these results suggest that GM130 is part of a cis-Golgi matrix and has a role in maintaining cis-Golgi structure.     

A peripheral protein associated with the cis-Golgi network redistributes in the intermediate compartment upon brefeldin A treatment

Human autoantibodies offer unique tools for the study of cellular constituents since they usually recognize highly conserved components, the most difficult to detect due to their low immunogenicity. The serum from a patient with Sjogren's syndrome (RM serum) showing a very high reactivity to the Golgi complex has been shown to immunoprecipitate and to immunodetect by Western blotting experiments a protein mol wt 210,000 (p210) that was shown to be peripheral and cytoplasmically disposed. A close examination of the p210 labeling revealed some differences with Golgi markers: RM serum staining was slightly more extensive than several Golgi markers and showed a discontinuous or granular appearance. Nocodazole induced a specific and early segregation of many p210-associated vesicles or tubules from Golgi apparatus. Upon brefeldin A treatment, p210 did not redistribute in the ER as did other Golgi proteins. In contrast, it exhibited a vesicular pattern reminiscent to that displayed by proteins residing in the intermediate compartment. Double staining immunofluorescence using the RM serum and the marker of the intermediate compartment, p58, revealed segregation of both proteins in control conditions but colocalization in BFA-treated cells. We have further demonstrated by combining different drug treatments that p210-containing elements in brefeldin A- treated cells belong indeed to the intermediate compartment. Experiments on brefeldin A recovery suggested that these p210 elements might play a role in reformation and repositioning of the Golgi apparatus. Ultrastructural localization performed by immunoperoxidase staining allowed us to establish that p210 interacted with the external side of an abundant tubulo-vesicular system on the cis side of the Golgi complex which extended to connecting structures and vesicles between saccules or stacks of cisternae, p210 appears to be a novel protein residing in the cis-Golgi network that may cycle between the Golgi apparatus and the intermediate compartment.     

Characterization of Grp1p,a novel cis-Golgi matrix protein

A high copy suppressor screen with sec34-2, a temperature-sensitive mutant defective in the late stages of ER to Golgi transport, has resulted in the identification of a novel gene called GRP1 (also called RUD3). GRP1 encodes a hydrophilic yeast protein related to the mammalian Golgi matrix protein golgin-160. A large portion of the protein is predicted to form a coiled-coil structure. Although GRP1 is not essential for growth, the loss of Grp1p results in a growth defect at high temperature. GRP1 genetically interacts with several genes involved in vesicle targeting/fusion stages of ER to Golgi transport. Despite these interactions, pulse chase analysis using Grp1p-depleted cells did not reveal a significant delay in the transit of the vacuolar protease carboxypeptidase Y. Grp1p-depleted cells efficiently secreted invertase which was underglycosylated, suggesting some disturbance of Golgi function. Grp1p-GFP predominantly colocalizes with the cis-Golgi marker Och1p. Despite lacking a signal peptide and a significant stretch of hydrophobic amino acids, Grp1p pellets with membranes. It is extracted with 1M NaCl or 0.1M Na(2)CO(3) (pH 11.0), but is surprisingly insoluble in 1% Triton X-100. Grp1p does not recycle to the ER when forward transport is blocked and a cis-Golgi marker (Och1p-HA), but not a trans-Golgi marker (Chs5p-HA), became dispersed in grp1 Delta cells after 1.5h incubation at 38.5 degrees C. Together, these data suggest that Grp1p is a novel matrix protein that is involved in the structural organization of the cis-Golgi.     

The mitotic phosphorylation cycle of the cis-Golgi matrix protein GM130

The cis-Golgi matrix protein GM130 is phosphorylated in mitosis on serine 25. Phosphorylation inhibits binding to p115, a vesicle-tethering protein, and has been implicated as an important step in the mitotic Golgi fragmentation process. We have generated an antibody that specifically recognizes GM130 phosphorylated on serine 25, and used this antibody to study the temporal regulation of phosphorylation in vivo. GM130 is phosphorylated in prophase as the Golgi complex starts to break down, and remains phosphorylated during further breakdown and partitioning of the Golgi fragments in metaphase and anaphase. In telophase, GM130 is dephosphorylated as the Golgi fragments start to reassemble. The timing of phosphorylation and dephosphorylation correlates with the dissociation and reassociation of p115 with Golgi membranes. GM130 phosphorylation and p115 dissociation appear specific to mitosis, since they are not induced by several drugs that trigger nonmitotic Golgi fragmentation. The phosphatase responsible for dephosphorylation of mitotic GM130 was identified as PP2A. The active species was identified as heterotrimeric phosphatase containing the Balpha regulatory subunit, suggesting a role for this isoform in the reassembly of mitotic Golgi membranes at the end of mitosis.     

TRAPP, a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion.

We previously identified BET3 by its genetic interactions with BET1, a gene whose SNARE-like product acts in endoplasmic reticulum (ER)-to-Golgi transport. To gain insight into the function of Bet3p, we added three c-myc tags to its C-terminus and immunopurified this protein from a clarified detergent extract. Here we report that Bet3p is a member of a large complex ( approximately 800 kDa) that we call TRAPP (transport protein particle). We propose that TRAPP plays a key role in the targeting and/or fusion of ER-to-Golgi transport vesicles with their acceptor compartment. The localization of Bet3p to the cis-Golgi complex, as well as biochemical studies showing that Bet3p functions on this compartment, support this hypothesis. TRAPP contains at least nine other constituents, five of which have been identified and shown to be highly conserved novel proteins.     

Nitrogen availability mediates soil carbon cycling response to climate warming: A meta-analysis

Global climate warming may induce a positive feedback through increasing soil carbon (C) release to the atmosphere. Although warming can affect both C input to and output from soil, direct and convincing evidence illustrating that warming induces a net change in soil C is still lacking. We synthesized the results from field warming experiments at 165 sites across the globe and found that climate warming had no significant effect on soil C stock. On average, warming significantly increased root biomass and soil respiration, but warming effects on root biomass and soil respiration strongly depended on soil nitrogen (N) availability. Under high N availability (soil C:N ratio < 15), warming had no significant effect on root biomass, but promoted the coupling between effect sizes of root biomass and soil C stock. Under relative N limitation (soil C:N ratio > 15), warming significantly enhanced root biomass. However, the enhancement of root biomass did not induce a corresponding C accumulation in soil, possibly because warming promoted microbial CO₂ release that offset the increased root C input. Also, reactive N input alleviated warming-induced C loss from soil, but elevated atmospheric CO₂ or precipitation increase/reduction did not. Together, our findings indicate that the relative availability of soil C to N (i.e., soil C:N ratio) critically mediates warming effects on soil C dynamics, suggesting that its incorporation into C-climate models may improve the prediction of soil C cycling under future global warming scenarios.     

A cycling protein complex required for selective autophagy

Survival of environmental stress conditions requires the maintenance of cellular homeostasis. To preserve this balance, cells utilize a degradative mechanism known as autophagy. During this process, in response to starvation or other stresses, bulk cytoplasm is non-specifically sequestered within double-membrane vesicles and delivered to the lysosome/vacuole for subsequent degradation and recycling. The cytoplasm to vacuole targeting (Cvt) pathway is a type of specific autophagy, which occurs constitutively during growing conditions. Here, we examine three autophagy-related (Atg) proteins, Atg9, Atg23 and Atg27, which exhibit a unique localization pattern, residing both at the pre-autophagosomal structure (PAS) and other peripheral sites. These proteins colocalize, interact with one another in vivo, and form a functional complex. Furthermore, all three proteins cycle between the PAS and the other sites, and depend upon one another for this movement. Our data suggest that Atg9, Atg23 and Atg27 play a role in Atg protein retrieval from the PAS. In addition, Atg9 and Atg27 are the only known integral membrane Atg proteins involved in vesicle formation; a better understanding of their function may offer insight into the mechanism of membrane delivery to the PAS, the site of double-membrane vesicle assembly.     

ULK1 phosphorylates Sec23A and mediates autophagy-induced inhibition of ER-to-Golgi traffic

Background

Autophagy is an inducible autodigestive process that allows cells to recycle proteins and other materials for survival during stress and nutrient deprived conditions. The kinase ULK1 is required to activate this process. ULK1 phosphorylates a number of target proteins and regulates many cellular processes including the early secretory pathway. Recently, ULK1 has been demonstrated to phosphorylate Sec16 and affects the transport of serotonin transporter at the ER exit sites (ERES), but whether ULK1 may affect the transport of other cargo proteins and general secretion has not been fully addressed.

Results

In this study, we identified Sec23A, a component of the COPII vesicle coat, as a target of ULK1 phosphorylation. Elevated autophagy, induced by amino acid starvation, rapamycin, or overexpression of ULK1 caused aggregation of the ERES, a region of the ER dedicated for the budding of COPII vesicles. Transport of cargo proteins was also inhibited under these conditions and was retained at the ERES. ULK1 phosphorylation of Sec23A reduced the interaction between Sec23A and Sec31A. We identified serine 207, serine 312 and threonine 405 on Sec23A as ULK1 phosphorylation sites. Among these residues, serine 207, when changed to phospho-deficient and phospho-mimicking mutants, most faithfully recapitulated the above-mentioned effects of ULK1 phospho-regulation.

Conclusion

These findings identify Sec23A as a new target of ULK1 and uncover a mechanism of coordinating intracellular protein transport and autophagy.

     

Targeting of protein ERGIC-53 to the ER/ERGIC/cis-Golgi recycling pathway

ERGIC-53 is a lectin-type membrane protein that continuously recycles between the ER, ER-Golgi intermediate compartment (ERGIC) and the cis- Golgi. To identify the targeting signals that mediate this recycling, N- glycosylated and myc-tagged variants of ERGIC-53 were constructed. By monitoring endoglycosidase H resistance, we measured the loss from the ER-ERGIC-cis-Golgi cycle of ERGIC-53. A domain exchange approach with the plasma membrane reporter protein CD4 showed that the transmembrane and the lumenal domains are not sufficient, while the cytoplasmic domain of ERGIC-53 is required and sufficient for pre-medial-Golgi localization. However, the ERGIC-53 cytoplasmic domain on CD4 lead to increased ER-staining by immunofluorescence microscopy indicating that this domain alone cannot provide for unbiased recycling through the ER- ERGIC-cis-Golgi compartments. Complete progress through the ER-ERGIC- cis-Golgi recycling pathway requires the cytoplasmic domain acting together with the lumenal domain of ERGIC-53. Dissection of the cytoplasmic domain revealed a COOH-terminal di-lysine ER-retrieval signal, KKFF, and an RSQQE targeting determinant adjacent to the transmembrane domain. Surprisingly, the two COOH-terminal phenylalanines influence the targeting. They reduce the ER-retrieval capacity of the di-lysine signal and modulate the RSQQE determinant.     

Surfactant protein A mediates pulmonary clearance of Staphylococcus aureus

Surfactant protein A has been shown to enhance opsonization and clearance of Staphylococcus aureus in vitro. Here, the phagocytosis of alveolar S. aureus was investigated in vivo using intravital microscopy. Fluorescence labelled S. aureus Newman cells were intratracheally administered to anesthetized mice and the alveolar surface was observed for fifteen minutes. Confirming previously reported in vitro data, surfactant protein A-deficient mice showed a significantly reduced uptake of bacteria compared to wild-type mice.     

Antibodies to rat pancreas Golgi subfractions: identification of a 58- kD cis-Golgi protein

A 58-kD cis-Golgi protein has been identified by generating polyclonal antibodies against heavy (cis) Golgi subfractions. Total microsomes isolated from rat pancreatic homogenates were subfractionated to yield a rough microsomal fraction (B1) and three smooth membrane subfractions (B2-B4) enriched in cis-, middle, and trans-Golgi elements, respectively. The heavy (cis) subfraction, B2 (d = 1.17 g/ml), was fractionated by Triton X-114 phase separation, and the proteins recovered in the detergent phase were used to immunize rabbits. One of the anti-B2 antibodies obtained gave a "Golgi"-staining pattern when screened by immunofluorescence on normal rat kidney cells and mouse RPC 5.4 myeloma cells. In rat pancreatic exocrine cells the antibody reacted with the plasmalemma as well as elements in the Golgi region. By immunoelectron microscopy, the antigen recognized by anti-B2 IgG was found to be restricted to cis-Golgi elements in myeloma cells where it was concentrated in the fenestrated cis-most cisterna and in some of the tubules and vesicles located along the cis face of the Golgi complex. By immunoprecipitation and immunoblotting, the anti-B2 IgG exclusively recognized a 58-kD protein in myeloma cells. The anti-B2 IgG reacted with several proteins in solubilized pancreatic B2 membranes, including a 58-kD protein, but affinity-purified anti-58-kD IgG reacted exclusively with the 58-kD protein. These results suggest that the 58-kD protein is a specific component of cis-Golgi membranes.     

Peroxin 5: A cycling receptor for protein translocation into peroxisomes

Peroxins are proteins that regulate the biogenesis of peroxisomes—small vesicular subcellular organelles essential for human life and health. A key peroxin – to date the best studied – is peroxin 5. Structurally, peroxin 5 is a bi-domain protein of about 70 kDa containing both globular and non-globular segments and displaying conformational flexibility. Functionally, it is a cycling receptor for importing essential enzymes into the peroxisome lumen, facilitated by highly promiscuous interactions with numerous proteins and possibly lipids. Peroxin 5 has medical significance in that (i) congenital defects can lead to fatal peroxisome biogenesis disorders, (ii) inefficient peroxisome targeting is linked to disease and aging and (iii) differences between human peroxin 5 and homologues in pathogens may be exploited in the development of therapeutics.     

A Dnmt2-like protein mediates DNA methylation in Drosophila

The methylation status of Drosophila DNA has been discussed controversially over a long time. Recent evidence has provided strong support for the existence of 5-methylcytosine in DNA preparations from embryonic stages of fly development. The Drosophila genome contains a single candidate DNA methyltransferase gene that has been termed Dnmt2. This gene belongs to a widely conserved family of putative DNA methyltransferases. However, no catalytic activity has been demonstrated for any Dnmt2-like protein yet. We have now established a protocol for the immunological detection of methylated cytosine in fly embryos. Confocal analysis of immunostained embryos provided direct evidence for the methylation of embryonic DNA. In order to analyse the function of Dnmt2 in DNA methylation, we depleted the protein by RNA interference. Depletion of Dnmt2 had no detectable effect on embryonic development and resulted in a complete loss of DNA methylation. Consistently, overexpression of Dnmt2 from an inducible transgene resulted in significant genomic hypermethylation at CpT and CpA dinucleotides. These results demonstrate that Dnmt2 is both necessary and sufficient for DNA methylation in Drosophila and suggest a novel CpT/A-specific DNA methyltransferase activity for Dnmt2 proteins.     

NADPH oxidase subunit 4 mediates cycling hypoxia-promoted radiation resistance in glioblastoma multiforme

Cycling hypoxia is a well-recognized phenomenon within animal and human solid tumors. It mediates tumor progression and radiotherapy resistance through mechanisms that involve reactive oxygen species (ROS) production. However, details of the mechanism underlying cycling hypoxia-mediated radioresistance remain obscure. We have previously shown that in glioblastoma, NADPH oxidase subunit 4 (Nox4) is a critical mediator involved in cycling hypoxia-mediated ROS production and tumor progression. Here, we examined the impact of an in vivo tumor microenvironment on Nox4 expression pattern and its impact on radiosensitivity in GBM8401 and U251, two glioblastoma cell lines stably transfected with a dual hypoxia-inducible factor-1 (HIF-1) signaling reporter construct. Furthermore, in order to isolate hypoxic tumor cell subpopulations from human glioblastoma xenografts based on the physiological and molecular characteristics of tumor hypoxia, several techniques were utilized. In this study, the perfusion marker Hoechst 33342 staining and HIF-1 activation labeling were used together with immunofluorescence imaging and fluorescence-activated cell sorting (FACS). Our results revealed that Nox4 was predominantly highly expressed in the endogenous cycling hypoxic areas with HIF-1 activation and blood perfusion within the solid tumor microenvironment. Moreover, when compared to the normoxic or chronic hypoxic cells, the cycling hypoxic tumor cells derived from glioblastoma xenografts have much higher Nox4 expression, ROS levels, and radioresistance. Nox4 suppression in intracerebral glioblastoma-bearing mice suppressed tumor microenvironment-mediated radioresistance and enhanced the efficiency of radiotherapy. In summary, our findings indicated that cycling hypoxia-induced Nox4 plays an important role in tumor microenvironment-promoted radioresistance in glioblastoma; hence, targeting Nox4 may be an attractive therapeutic strategy for blocking cycling hypoxia-mediated radioresistance.     

ZFPL1, a novel ring finger protein required for cis-Golgi integrity and efficient ER-to-Golgi transport

The Golgi apparatus occupies a central position within the secretory pathway, but the molecular mechanisms responsible for its assembly and organization remain poorly understood. We report here the identification of zinc finger protein-like 1 (ZFPL1) as a novel structural component of the Golgi apparatus. ZFPL1 is a conserved and widely expressed integral membrane protein with two predicted zinc fingers at the N-terminus, the second of which is a likely ring domain. ZFPL1 directly interacts with the cis-Golgi matrix protein GM130. Depletion of ZFPL1 results in the accumulation of cis-Golgi matrix proteins in the intermediate compartment (IC) and the tubulation of cis-Golgi and IC membranes. Loss of ZFPL1 function also impairs cis-Golgi assembly following brefeldin A washout and slows the rate of cargo trafficking into the Golgi apparatus. Effects upon Golgi matrix protein localization and cis-Golgi structure can be rescued by wild-type ZFPL1 but not mutants defective in GM130 binding. Together, these data suggest that ZFPL1 has an important function in maintaining the integrity of the cis-Golgi and that it does so through interactions with GM130.     

A single peroxisomal targeting signal mediates matrix protein import in diatoms

Peroxisomes are single membrane bound compartments. They are thought to be present in almost all eukaryotic cells, although the bulk of our knowledge about peroxisomes has been generated from only a handful of model organisms. Peroxisomal matrix proteins are synthesized cytosolically and posttranslationally imported into the peroxisomal matrix. The import is generally thought to be mediated by two different targeting signals. These are respectively recognized by the two import receptor proteins Pex5 and Pex7, which facilitate transport across the peroxisomal membrane. Here, we show the first in vivo localization studies of peroxisomes in a representative organism of the ecologically relevant group of diatoms using fluorescence and transmission electron microscopy. By expression of various homologous and heterologous fusion proteins we demonstrate that targeting of Phaeodactylum tricornutum peroxisomal matrix proteins is mediated only by PTS1 targeting signals, also for proteins that are in other systems imported via a PTS2 mode of action. Additional in silico analyses suggest this surprising finding may also apply to further diatoms. Our data suggest that loss of the PTS2 peroxisomal import signal is not reserved to Caenorhabditis elegans as a single exception, but has also occurred in evolutionary divergent organisms. Obviously, targeting switching from PTS2 to PTS1 across different major eukaryotic groups might have occurred for different reasons. Thus, our findings question the widespread assumption that import of peroxisomal matrix proteins is generally mediated by two different targeting signals. Our results implicate that there apparently must have been an event causing the loss of one targeting signal even in the group of diatoms. Different possibilities are discussed that indicate multiple reasons for the detected targeting switching from PTS2 to PTS1.