scholarly journals The Golgi-Associated Hook3 Protein Is a Member of a Novel Family of Microtubule-Binding Proteins

2001 ◽  
Vol 152 (5) ◽  
pp. 923-934 ◽  
Author(s):  
Jason H. Walenta ◽  
Aaron J. Didier ◽  
Xinran Liu ◽  
Helmut Krämer
Keyword(s):  
Golgi Complex ◽  
Membrane Trafficking ◽  
Spatial Organization ◽  
Large Fraction ◽  
Coiled Coil ◽  
Brefeldin A ◽  
Associated Proteins ◽  
Terminal Domains

Microtubules are central to the spatial organization of diverse membrane-trafficking systems. Here, we report that Hook proteins constitute a novel family of cytosolic coiled coil proteins that bind to organelles and to microtubules. The conserved NH2-terminal domains of Hook proteins mediate attachment to microtubules, whereas the more divergent COOH-terminal domains mediate the binding to organelles. Human Hook3 bound to Golgi membranes in vitro and was enriched in the cis-Golgi in vivo. Unlike other cis-Golgi–associated proteins, however, a large fraction of Hook3 maintained its juxtanuclear localization after Brefeldin A treatment, indicating a Golgi-independent mechanism for Hook3 localization. Because overexpression of Hook3 caused fragmentation of the Golgi complex, we propose that Hook3 participates in defining the architecture and localization of the mammalian Golgi complex.

scholarly journals Lava Lamp, a Novel Peripheral Golgi Protein, Is Required for Drosophila melanogaster Cellularization

2000 ◽  
Vol 151 (4) ◽  
pp. 905-918 ◽  
Author(s):  
John C. Sisson ◽  
Christine Field ◽  
Richard Ventura ◽  
Anne Royou ◽  
William Sullivan
Keyword(s):  
Membrane Trafficking ◽  
Biochemical Analysis ◽  
Potent Inhibitor ◽  
Animal Cell ◽  
Coiled Coil ◽  
Brefeldin A ◽  
Microtubule Associated Proteins ◽  
Golgi Bodies ◽  
Associated Proteins ◽  
Golgi Protein

Drosophila cellularization and animal cell cytokinesis rely on the coordinated functions of the microfilament and microtubule cytoskeletal systems. To identify new proteins involved in cellularization and cytokinesis, we have conducted a biochemical screen for microfilament/microtubule-associated proteins (MMAPs). 17 MMAPs were identified; seven have been previously implicated in cellularization and/or cytokinesis, including KLP3A, Anillin, Septins, and Dynamin. We now show that a novel MMAP, Lava Lamp (Lva), is also required for cellularization. Lva is a coiled-coil protein and, unlike other proteins previously implicated in cellularization or cytokinesis, it is Golgi associated. Our functional analysis shows that cellularization is dramatically inhibited upon injecting anti–Lva antibodies (IgG and Fab) into embryos. In addition, we show that brefeldin A, a potent inhibitor of membrane trafficking, also inhibits cellularization. Biochemical analysis demonstrates that Lva physically interacts with the MMAPs Spectrin and CLIP190. We suggest that Lva and Spectrin may form a Golgi-based scaffold that mediates the interaction of Golgi bodies with microtubules and facilitates Golgi-derived membrane secretion required for the formation of furrows during cellularization. Our results are consistent with the idea that animal cell cytokinesis depends on both actomyosin-based contraction and Golgi-derived membrane secretion.

scholarly journals Packing volume of sedimented microtubules: regulation and potential relationship to an intracellular matrix.

1985 ◽  
Vol 101 (4) ◽  
pp. 1492-1500 ◽  
Author(s):  
P A Brown ◽  
R D Berlin
Keyword(s):  
Theoretical Analysis ◽  
Specific Volume ◽  
Mechanical Stability ◽  
Large Fraction ◽  
Bovine Brain ◽  
Associated Proteins ◽  
Irreducible Space ◽  
Specific Volumes

To determine the contribution of microtubules to a hypothetical intracellular matrix, we have analyzed the space occupied by microtubules in vitro. Taxol-stabilized microtubules assembled from purified (three-times-cycled) bovine brain microtubule protein were pelleted by centrifugation under standardized conditions. The specific volume of the pellet, defined as the microliter volume per milligram protein, was 22.4. As suggested by others, this volume was strongly dependent on microtubule-associated proteins (MAPs), as shown by quantitation of the effects of purified MAP supplementation on specific volume. The specific volumes of microtubule pellets stripped of MAPs by high salt or chymotryptic digestion approached the mathematically optimal (least occupied space) and increased 14-fold with the highest MAP concentrations employed. Packing was also dependent on pH. Specific volumes comparable to those of MAP-depleted microtubules were attainable at pH's from 5.5 to 6.0, and specific volumes more than doubled at pH 7.5. MAP content was unaffected by pH. We present a theoretical analysis that suggests that as microtubules are centrifuged the mixture behaves as a liquid crystal. With packing, the mixture undergoes an isotropic-nematic phase transition in which the microtubules become oriented principally as parallel rods, mimicking their orientation in vivo. From the known concentration of microtubules in vivo, it can be inferred from our measurements that in some cells a large fraction, perhaps 40-50% of the cytosolic volume, is occupied by microtubules that form a mechanically irreducible space. Further theoretical analysis employing Ogston's formulation of the penetrability of fibrous networks suggests that the space between microtubules (in contrast to the extracellular matrix) imposes little barrier to the diffusion of macromolecules. A microtubule array thus achieves mechanical stability without affecting transport by diffusion. The space can accommodate other fibrous networks that could then affect transport, and, as we show, the space itself may be regulated by MAP content and intracellular pH.

scholarly journals Organization of the Yeast Golgi Complex into at Least Four Funtionally Distinct Compartments

2000 ◽  
Vol 11 (1) ◽  
pp. 171-182 ◽  
Author(s):  
William T. Brigance ◽  
Charles Barlowe ◽  
Todd R. Graham
Keyword(s):  
Golgi Complex ◽  
Brefeldin A ◽  
Proteolytic Processing ◽  
Golgi Compartment ◽  
Specific Antisera ◽  
Sensitive Factor ◽  
Processing Event ◽  
Golgi Network

Pro-α-factor (pro-αf) is posttranslationally modified in the yeast Golgi complex by the addition of α1,6-, α1,2-, and α1,3-linked mannose to N-linked oligosaccharides and by a Kex2p-initiated proteolytic processing event. Previous work has indicated that the α1,6- and α1,3-mannosylation and Kex2p-dependent processing of pro-αf are initiated in three distinct compartments of the Golgi complex. Here, we present evidence that α1,2-mannosylation of pro-αf is also initiated in a distinct Golgi compartment. Linkage-specific antisera and an endo-α1,6-d-mannanase (endoM) were used to quantitate the amount of each pro-αf intermediate during transport through the Golgi complex. We found that α1,6-, α1,2-, and α1,3-mannose were sequentially added to pro-αf in a temporally ordered manner, and that the intercompartmental transport factor Sec18p/N-ethylmaleimide-sensitive factor was required for each step. The Sec18p dependence implies that a transport event was required between each modification event. In addition, most of the Golgi-modified pro-αf that accumulated in brefeldin A-treated cells received only α1,6-mannosylation as did ∼50% of pro-αf transported to the Golgi in vitro. This further supports the presence of an early Golgi compartment that houses an α1,6-mannosyltransferase but lacks α1,2-mannosyltransferase activity in vivo. We propose that the α1,6-, α1,2-, and α1,3-mannosylation and Kex2p-dependent processing events mark the cis, medial,trans, and trans-Golgi network of the yeast Golgi complex, respectively.

scholarly journals ADP-Ribosylation Factor 1 (ARF1) Regulates Recruitment of the AP-3 Adaptor Complex to Membranes

1998 ◽  
Vol 142 (2) ◽  
pp. 391-402 ◽  
Author(s):  
Chean Eng Ooi ◽  
Esteban C. Dell'Angelica ◽  
Juan S. Bonifacino
Keyword(s):  
Membrane Trafficking ◽  
Brefeldin A ◽  
Membrane Association ◽  
Coat Proteins ◽  
Nucleotide Exchange ◽  
Membrane Recruitment ◽  
Adp Ribosylation ◽  
Adp Ribosylation Factor

Small GTP-binding proteins such as ADP- ribosylation factor 1 (ARF1) and Sar1p regulate the membrane association of coat proteins involved in intracellular membrane trafficking. ARF1 controls the clathrin coat adaptor AP-1 and the nonclathrin coat COPI, whereas Sar1p controls the nonclathrin coat COPII. In this study, we demonstrate that membrane association of the recently described AP-3 adaptor is regulated by ARF1. Association of AP-3 with membranes in vitro was enhanced by GTPγS and inhibited by brefeldin A (BFA), an inhibitor of ARF1 guanine nucleotide exchange. In addition, recombinant myristoylated ARF1 promoted association of AP-3 with membranes. The role of ARF1 in vivo was examined by assessing AP-3 subcellular localization when the intracellular level of ARF1-GTP was altered through overexpression of dominant ARF1 mutants or ARF1- GTPase-activating protein (GAP). Lowering ARF1-GTP levels resulted in redistribution of AP-3 from punctate membrane-bound structures to the cytosol as seen by immunofluorescence microscopy. In contrast, increasing ARF1-GTP levels prevented redistribution of AP-3 to the cytosol induced by BFA or energy depletion. Similar experiments with mutants of ARF5 and ARF6 showed that these other ARF family members had little or no effect on AP-3. Taken together, our results indicate that membrane recruitment of AP-3 is promoted by ARF1-GTP. This finding suggests that ARF1 is not a regulator of specific coat proteins, but rather is a ubiquitous molecular switch that acts as a transducer of diverse signals influencing coat assembly.

scholarly journals Molecular Cloning and Characterization of Phocein, a Protein Found from the Golgi Complex to Dendritic Spines

2001 ◽  
Vol 12 (3) ◽  
pp. 663-673 ◽  
Author(s):  
Gilbert Baillat ◽  
Abdelaziz Moqrich ◽  
Francis Castets ◽  
Agnès Baude ◽  
Yannick Bailly ◽  
...  
Keyword(s):  
Golgi Complex ◽  
Hela Cells ◽  
Brefeldin A ◽  
Calmodulin Binding ◽  
Adult Rat ◽  
Adult Rat Brain ◽  
Clathrin Adaptor

Phocein is a widely expressed, highly conserved intracellular protein of 225 amino acids, the sequence of which has limited homology to the ς subunits from clathrin adaptor complexes and contains an additional stretch bearing a putative SH3-binding domain. This sequence is evolutionarily very conserved (80% identity betweenDrosophila melanogaster and human). Phocein was discovered by a yeast two-hybrid screen using striatin as a bait. Striatin, SG2NA, and zinedin, the three mammalian members of the striatin family, are multimodular, WD-repeat, and calmodulin-binding proteins. The interaction of phocein with striatin, SG2NA, and zinedin was validated in vitro by coimmunoprecipitation and pull-down experiments. Fractionation of brain and HeLa cells showed that phocein is associated with membranes, as well as present in the cytosol where it behaves as a protein complex. The molecular interaction between SG2NA and phocein was confirmed by their in vivo colocalization, as observed in HeLa cells where antibodies directed against either phocein or SG2NA immunostained the Golgi complex. A 2-min brefeldin A treatment of HeLa cells induced the redistribution of both proteins. Immunocytochemical studies of adult rat brain sections showed that phocein reactivity, present in many types of neurons, is strictly somato-dendritic and extends down to spines, just as do striatin and SG2NA.

scholarly journals Transmission of cell stress from endoplasmic reticulum to mitochondria

2002 ◽  
Vol 157 (7) ◽  
pp. 1151-1160 ◽  
Author(s):  
Osamu Hori ◽  
Fusae Ichinoda ◽  
Takashi Tamatani ◽  
Atsushi Yamaguchi ◽  
Naoya Sato ◽  
...  
Keyword(s):  
Er Stress ◽  
Nuclear Gene ◽  
Brefeldin A ◽  
Wild Type ◽  
Nuclear Gene Expression ◽  
Cultured Astrocytes ◽  
Associated Proteins ◽  
Steady State Levels

The rat homologue of a mitochondrial ATP-dependent protease Lon was cloned from cultured astrocytes exposed to hypoxia. Expression of Lon was enhanced in vitro by hypoxia or ER stress, and in vivo by brain ischemia. These observations suggested that changes in nuclear gene expression (Lon) triggered by ER stress had the potential to impact important mitochondrial processes such as assembly and/or degradation of cytochrome c oxidase (COX). In fact, steady-state levels of nuclear-encoded COX IV and V were reduced, and mitochondrial-encoded subunit II was rapidly degraded under ER stress. Treatment of cells with cycloheximide caused a similar imbalance in the accumulation of COX subunits, and enhanced mRNA for Lon and Yme1, the latter another mitochondrial ATP-dependent protease. Furthermore, induction of Lon or GRP75/mtHSP70 by ER stress was inhibited in PERK (−/−) cells. Transfection studies revealed that overexpression of wild-type or proteolytically inactive Lon promoted assembly of COX II into a COX I–containing complex, and partially prevented mitochondrial dysfunction caused by brefeldin A or hypoxia. These observations demonstrated that suppression of protein synthesis due to ER stress has a complex effect on the synthesis of mitochondrial-associated proteins, both COX subunits and ATP-dependent proteases and/or chaperones contributing to assembly of the COX complex.

scholarly journals Dissociation of Coatomer from Membranes Is Required for Brefeldin A–induced Transfer of Golgi Enzymes to the Endoplasmic Reticulum

1997 ◽  
Vol 137 (2) ◽  
pp. 319-333 ◽  
Author(s):  
Jochen Scheel ◽  
Rainer Pepperkok ◽  
Martin Lowe ◽  
Gareth Griffiths ◽  
Thomas E. Kreis
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Transport ◽  
Golgi Complex ◽  
Mammalian Cells ◽  
Brefeldin A ◽  
Retrograde Transport ◽  
Marker Proteins ◽  
Kdel Receptor

Addition of brefeldin A (BFA) to mammalian cells rapidly results in the removal of coatomer from membranes and subsequent delivery of Golgi enzymes to the endoplasmic reticulum (ER). Microinjected anti-EAGE (intact IgG or Fab-fragments), antibodies against the “EAGE”-peptide of β-COP, inhibit BFA-induced redistribution of β-COP in vivo and block transfer of resident proteins of the Golgi complex to the ER; tubulo-vesicular clusters accumulate and Golgi membrane proteins concentrate in cytoplasmic patches containing β-COP. These patches are devoid of marker proteins of the ER, the intermediate compartment (IC), and do not contain KDEL receptor. Interestingly, relocation of KDEL receptor to the IC, where it colocalizes with ERGIC53 and ts-O45-G, is not inhibited under these conditions. While no stacked Golgi cisternae remain in these injected cells, reassembly of stacks of Golgi cisternae following BFA wash-out is inhibited to only ∼50%. Mono- or divalent anti-EAGE stabilize binding of coatomer to membranes in vitro, at least as efficiently as GTPγS. Taken together these results suggest that enhanced binding of coatomer to membranes completely inhibits the BFA-induced retrograde transport of Golgi resident proteins to the ER, probably by inhibiting fusion of Golgi with ER membranes, but does not interfere with the disassembly of the stacked Golgi cisternae and recycling of KDEL receptor to the IC. These results confirm our previous results suggesting that COPI is involved in anterograde membrane transport from the ER/IC to the Golgi complex (Pepperkok et al., 1993), and corroborate that COPI regulates retrograde membrane transport between the Golgi complex and ER in mammalian cells.

scholarly journals Phasins, Multifaceted Polyhydroxyalkanoate Granule-Associated Proteins

2016 ◽  
Vol 82 (17) ◽  
pp. 5060-5067 ◽  
Author(s):  
Mariela P. Mezzina ◽  
M. Julia Pettinari
Keyword(s):  
Binding Capacity ◽  
Coiled Coil ◽  
Active Role ◽  
Structural Features ◽  
Structural Function ◽  
Associated Proteins ◽  
Α Helix ◽  
Pha Granule

ABSTRACTPhasins are the major polyhydroxyalkanoate (PHA) granule-associated proteins. They promote bacterial growth and PHA synthesis and affect the number, size, and distribution of the granules. These proteins can be classified in 4 families with distinctive characteristics. Low-resolution structural studies andin silicopredictions were performed in order to elucidate the structure of different phasins. Most of these proteins share some common structural features, such as a preponderant α-helix composition, the presence of disordered regions that provide flexibility to the protein, and coiled-coil interacting regions that form oligomerization domains. Due to their amphiphilic nature, these proteins play an important structural function, forming an interphase between the hydrophobic content of PHA granules and the hydrophilic cytoplasm content. Phasins have been observed to affect both PHA accumulation and utilization. Apart from their role as granule structural proteins, phasins have a remarkable variety of additional functions. Different phasins have been determined to (i) activate PHA depolymerization, (ii) increase the expression and activity of PHA synthases, (iii) participate in PHA granule segregation, and (iv) have bothin vivoandin vitrochaperone activities. These properties suggest that phasins might play an active role in PHA-related stress protection and fitness enhancement. Due to their granule binding capacity and structural flexibility, several biotechnological applications have been developed using different phasins, increasing the interest in the study of these remarkable proteins.

scholarly journals Extensive phosphorylation of AMPA receptors in neurons

2016 ◽  
Vol 113 (33) ◽  
pp. E4920-E4927 ◽  
Author(s):  
Graham H. Diering ◽  
Seok Heo ◽  
Natasha K. Hussain ◽  
Bian Liu ◽  
Richard L. Huganir
Keyword(s):  
Synaptic Plasticity ◽  
Protein Interactions ◽  
Membrane Trafficking ◽  
Ampa Receptors ◽  
Posttranslational Modifications ◽  
Large Fraction ◽  
Large Body ◽  
Long Term Potentiation

Regulation of AMPA receptor (AMPAR) function is a fundamental mechanism controlling synaptic strength during long-term potentiation/depression and homeostatic scaling. AMPAR function and membrane trafficking is controlled by protein–protein interactions, as well as by posttranslational modifications. Phosphorylation of the GluA1 AMPAR subunit at S845 and S831 play especially important roles during synaptic plasticity. Recent controversy has emerged regarding the extent to which GluA1 phosphorylation may contribute to synaptic plasticity. Here we used a variety of methods to measure the population of phosphorylated GluA1-containing AMPARs in cultured primary neurons and mouse forebrain. Phosphorylated GluA1 represents large fractions from 12% to 50% of the total population under basal and stimulated conditions in vitro and in vivo. Furthermore, a large fraction of synapses are positive for phospho-GluA1–containing AMPARs. Our results support the large body of research indicating a prominent role of GluA1 phosphorylation in synaptic plasticity.

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