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1.
Biochem Soc Trans ; 29(Pt 4): 461-7, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11498009

ABSTRACT

Accurate data on the three-dimensional architecture of the Golgi is prerequisite for evaluating the mechanisms of transit through this organelle. Here we detail the structure of the Golgi ribbon within part of an insulin-secreting cell in three dimensions at approximately 6 nm resolution. Rapid freezing, freeze-substitution and electron tomography were employed. The Golgi in this region is composed of seven cisternae. The cis-most element is structurally intermediate between the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) and the cis-most cisterna characterized in three dimensions at high resolution in a normal rat kidney cell [Ladinsky, Mastronarde, McIntosh, Howell and Staehelin (1999) J. Cell Biol. 144, 1135-1149]. There are three trans-cisternae that demonstrate morphological and functional variation. The membrane surface areas and volumes of these elements decrease from cis to trans. The two trans-most cisternae are dissociated from the stack and are fragmented by tubulation. ER closely adheres to and inserts between individual trans-cisternae. Many of the 2119 small, clathrin-negative vesicles that are in close proximity to the Golgi fill the region where trans-cisternae have moved out of register with the ribbon. These data provide evidence that cisternal progression/maturation, trafficking via membrane tubules and vesicle-mediated transport act in concert in the same region of the Golgi ribbon, and suggest an important role for the ER in regulating membrane dynamics at the trans-Golgi.


Subject(s)
Golgi Apparatus/physiology , Islets of Langerhans/physiology , Signal Transduction/physiology , Animals , Biological Transport , Cell Fractionation , Cell Line , Islets of Langerhans/ultrastructure , Microscopy, Electron , Models, Structural , trans-Golgi Network/physiology , trans-Golgi Network/ultrastructure
2.
Proc Natl Acad Sci U S A ; 98(5): 2399-406, 2001 Feb 27.
Article in English | MEDLINE | ID: mdl-11226251

ABSTRACT

The positional relationships among all of the visible organelles in a densely packed region of cytoplasm from an insulin secreting, cultured mammalian cell have been analyzed in three dimensions (3-D) at approximately 6 nm resolution. Part of a fast frozen/freeze-substituted HIT-T15 cell that included a large portion of the Golgi ribbon was reconstructed in 3-D by electron tomography. The reconstructed volume (3.1 x 3.2 x 1.2 microm(3)) allowed sites of interaction between organelles, and between microtubules and organellar membranes, to be accurately defined in 3-D and quantitatively analyzed by spatial density analyses. Our data confirm that the Golgi in an interphase mammalian cell is a single, ribbon-like organelle composed of stacks of flattened cisternae punctuated by openings of various sizes [Rambourg, A., Clermont, Y., & Hermo, L. (1979) Am. J. Anat. 154, 455-476]. The data also show that the endoplasmic reticulum (ER) is a single continuous compartment that forms close contacts with mitochondria, multiple trans Golgi cisternae, and compartments of the endo-lysosomal system. This ER traverses the Golgi ribbon from one side to the other via cisternal openings. Microtubules form close, non-random associations with the cis Golgi, the ER, and endo-lysosomal compartments. Despite the dense packing of organelles in this Golgi region, approximately 66% of the reconstructed volume is calculated to represent cytoplasmic matrix. We relate the intimacy of structural associations between organelles in the Golgi region, as quantified by spatial density analyses, to biochemical mechanisms for membrane trafficking and organellar communication in mammalian cells.


Subject(s)
Golgi Apparatus/ultrastructure , Islets of Langerhans/ultrastructure , Organelles/ultrastructure , Tomography/methods , Cell Line , Electrons , Models, Biological
3.
Electrophoresis ; 21(16): 3441-59, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11079564

ABSTRACT

The discovery of novel proteins resident to the Golgi complex will fuel our future studies of Golgi structure/function and provide justification for proteomic analysis of this organelle. Our approach to Golgi proteomics was to first isolate and characterize the intact organelle free of proteins in transit by use of tissue pretreated with cycloheximide. Then the stacked Golgi fraction was fractionated into biochemically defined subfractions: Triton X-114 insoluble, aqueous, and detergent phases. The aqueous and detergent phases were further fractionated by anion-exchange column chromatography. In addition, radiolabeled cytosol was incubated with stacked Golgi fractions containing proteins in transit, and the proteins bound to the Golgi stacks in an energy-dependent manner were characterized. All fractions were analyzed by two-dimensional (2-D) gel electrophoresis and identification numbers were given to 588 unique 2-D spots. Tandem mass spectrometry was used to analyze 93 of the most abundant 2-D spots taken from preparative Triton X-114 insoluble, aqueous and detergent phase 2-D gels. Fifty-one known and 22 unknown proteins were identified. This study represents the first installment in the mammalian Golgi proteome database. Our data suggest that cell fractionation followed by biochemical dissection of specific classes of molecules provides a significant advantage for the identification of low abundance proteins in organelles.


Subject(s)
Golgi Apparatus/metabolism , Liver/metabolism , Proteins/isolation & purification , Proteome , Animals , Chromatography, High Pressure Liquid , Electrophoresis, Gel, Two-Dimensional , Liver/ultrastructure , Mass Spectrometry , Octoxynol , Polyethylene Glycols , Proteins/chemistry , Rats , Silver Staining
4.
Electrophoresis ; 21(16): 3470-82, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11079566

ABSTRACT

The synthesis and secretion of lipids by mammary epithelial cells is a highly ordered process that involves several distinct steps. Triacylglycerols are synthesized in the endoplasmic reticulum and incorporated into microlipid droplets which coalesce into cytoplasmic lipid droplets. These are vectorially transported to the apical plasma membrane where they are secreted into the milk surrounded by a membrane bilayer. The origin of this membrane as well as the mechanism by which cytoplasmic lipid droplets form and become surrounded by membrane is poorly understood. Proteomic analysis of the protein composition of milk fat globules and cytoplasmic lipid droplet has revealed that the endoplasmic reticulum is not only involved in the synthesis of the lipid but also potentially contributes to the membrane component of milk fat globules. The proteins identified suggest possible mechanisms of multiple steps during this process. Completion of the proteome of milk fat globule membranes and cytoplasmic lipid droplets will provide the necessary reporter molecules to follow and dissect the mechanisms of the sorting and ultimate secretion of cytoplasmic lipid droplets.


Subject(s)
Endoplasmic Reticulum/metabolism , Lipid Metabolism , Mammary Glands, Animal/metabolism , Proteome , Animals , Electrophoresis, Gel, Two-Dimensional , Epithelial Cells/metabolism , Epithelial Cells/ultrastructure , Female , Lactation , Liver/metabolism , Liver/ultrastructure , Mammary Glands, Animal/ultrastructure , Mass Spectrometry , Mice , Microscopy, Electron
5.
Biophys J ; 78(6): 2918-28, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10827972

ABSTRACT

The Golgi complex is present in every eukaryotic cell and functions in posttranslational modifications and sorting of proteins and lipids to post-Golgi destinations. Both functions require an acidic lumenal pH and transport of substrates into and by-products out of the Golgi lumen. Endogenous ion channels are expected to be important for these features, but none has been described. Ion channels from an enriched Golgi fraction cleared of transiting proteins were incorporated into planar lipid bilayers. Eighty percent of the single-channel recordings revealed the same anion channel. This channel has novel properties and has been named GOLAC (Golgi anion channel). The channel has six subconductance states with a maximum conductance of 130 pS, is open over 95% of the time, and is not voltage-gated. Significant for Golgi function, the channel conductance is increased by reduction of pH on the lumenal surface. This channel may serve two nonexclusive functions: providing counterions for the acidification of the Golgi lumen by the H(+)-ATPase and removal of inorganic phosphate generated by glycosylation and sulfation of proteins and lipids in the Golgi.


Subject(s)
Golgi Apparatus/physiology , Ion Channels/physiology , Lipid Bilayers , 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/pharmacology , Animals , Chlorides/pharmacology , Golgi Apparatus/ultrastructure , Liver/ultrastructure , Membrane Potentials/drug effects , Phosphatidylethanolamines , Phosphatidylserines , Potassium Chloride/pharmacology , Rats
6.
Traffic ; 1(10): 769-82, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11208067

ABSTRACT

Organellar compartments involved in secretion are expanded during the transition from late pregnancy (basal secretory state) to lactation (maximal secretory state) to accommodate for the increased secretory function required for copious milk production in mammary epithelial cells. The Golgi complex is a major organelle of the secretory pathway and functions to sort, package, distribute, and post-translationally modify newly synthesized proteins and membrane lipids. These complex functions of the Golgi are reflected in the protein complement of the organelle. Therefore, using proteomics, the protein complements of Golgi fractions isolated at two functional states (basal and maximal) were compared to identify some of the molecular changes that occur during this transition. This global analysis has revealed that only a subset of the total proteins is upregulated from steady state during the transition. Identification of these proteins by tandem mass spectrometry has revealed several classes of proteins involved in the regulation of membrane fusion and secretion. This first installment of the functional proteomic analysis of the Golgi complex begins to define the molecular basis for the transition from basal to maximal secretion.


Subject(s)
Golgi Apparatus/metabolism , Mammary Glands, Animal/metabolism , Proteome , Animals , Cell Fractionation , Cycloheximide/pharmacology , Electrophoresis, Gel, Two-Dimensional , Epithelial Cells/metabolism , Epithelial Cells/ultrastructure , Female , Mammary Glands, Animal/ultrastructure , Mass Spectrometry , Microscopy, Electron , Pregnancy , Rats , Rats, Sprague-Dawley
7.
Traffic ; 1(12): 963-75, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11208086

ABSTRACT

The known functions of the Golgi complex include the sorting, packaging, post-translational modification, and transport of secretory proteins, membrane proteins, and lipids. Other functions still remain elusive to cell biologists. With the goal of identifying novel Golgi proteins, a proteomics project was undertaken to map the major proteins of the organelle using two-dimensional gels, to identify the unknowns using tandem mass spectrometry, and to screen for Golgi residents using GFP-fusion constructs. Multiple unknowns were identified, and the initial characterization of one of these proteins is reported here. GMx33 alpha is a member of a conserved family of cytosolic Golgi-associated proteins with no known homology to any known functional domain or protein. Biochemical analyses show that GMx33 alpha differentially partitions into all phases of multiple detergent extractions, and two-dimensional immunoblots reveal that there are multiple differentially modified forms of GMx33 alpha associated with the Golgi, several of which are phosphorylated. Evidence suggests that these post-translational modifications regulate its association with the Golgi. GMx33 alpha was not found on Golgi budded vesicles, and immuno-electron microscopy co-localizes GMx33 alpha to the trans-face on the same three cisternae as TGN38 in normal rat kidney cells. This work represents the preliminary characterization of a novel family of trans-Golgi-associated proteins.


Subject(s)
Carrier Proteins/chemistry , Glycoproteins , Membrane Proteins , Proteome/analysis , trans-Golgi Network/chemistry , Amino Acid Motifs , Amino Acid Sequence , Animals , Carrier Proteins/analysis , Cell Line , Cell-Free System , Cloning, Molecular , Detergents , Electrophoresis, Gel, Two-Dimensional , Genes, Reporter , Humans , Immunoblotting , Liver/chemistry , Membrane Glycoproteins/analysis , Molecular Sequence Data , Rats , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/chemistry , Sequence Alignment , trans-Golgi Network/metabolism
8.
Traffic ; 1(9): 713-23, 2000 Sep.
Article in English | MEDLINE | ID: mdl-11208159

ABSTRACT

TGN38 luminal domain (TGN38LD) was expressed in Cos-7 cells to identify potential binding partners. The luminal domain was secreted but, surprisingly, a significant portion bound to the plasma membrane. Cells overexpressing TGN38LD or the full-length molecule detached from the substratum and left footprints positive for TGN38. Unexpectedly, in these cells, TGN38 colocalizes with integrin alpha 5 beta 1 at the Golgi, the cell surface or in the footprints and an increased amount of both integrin subunits on the plasma membrane was observed. Under physiological conditions when TGN38 is not overexpressed, it interacts with integrin beta 1. This was demonstrated by reciprocal co-immunoprecipitation of integrin beta 1 and TGN38. Functional analysis reveals that modification of the trafficking of TGN38 results in a parallel change in the distribution of integrin alpha 5 beta 1, leading to the conclusion that TGN38 is involved in the trafficking of integrin beta 1.


Subject(s)
Cell Membrane/metabolism , Glycoproteins , Integrin beta1/metabolism , Membrane Glycoproteins/metabolism , Protein Transport/physiology , trans-Golgi Network/metabolism , Animals , COS Cells/metabolism , COS Cells/ultrastructure , Cell Adhesion/physiology , Cell Membrane/ultrastructure , Glycosylation , Intracellular Membranes/metabolism , Membrane Glycoproteins/chemistry , Membrane Proteins/metabolism , Protein Structure, Tertiary/physiology
9.
J Cell Biol ; 144(6): 1135-49, 1999 Mar 22.
Article in English | MEDLINE | ID: mdl-10087259

ABSTRACT

Three-dimensional reconstructions of portions of the Golgi complex from cryofixed, freeze-substituted normal rat kidney cells have been made by dual-axis, high-voltage EM tomography at approximately 7-nm resolution. The reconstruction shown here ( approximately 1 x 1 x 4 microm3) contains two stacks of seven cisternae separated by a noncompact region across which bridges connect some cisternae at equivalent levels, but none at nonequivalent levels. The rest of the noncompact region is filled with both vesicles and polymorphic membranous elements. All cisternae are fenestrated and display coated buds. They all have about the same surface area, but they differ in volume by as much as 50%. The trans-most cisterna produces exclusively clathrin-coated buds, whereas the others display only nonclathrin coated buds. This finding challenges traditional views of where sorting occurs within the Golgi complex. Tubules with budding profiles extend from the margins of both cis and trans cisternae. They pass beyond neighboring cisternae, suggesting that these tubules contribute to traffic to and/or from the Golgi. Vesicle-filled "wells" open to both the cis and lateral sides of the stacks. The stacks of cisternae are positioned between two types of ER, cis and trans. The cis ER lies adjacent to the ER-Golgi intermediate compartment, which consists of discrete polymorphic membranous elements layered in front of the cis-most Golgi cisterna. The extensive trans ER forms close contacts with the two trans-most cisternae; this apposition may permit direct transfer of lipids between ER and Golgi membranes. Within 0.2 microm of the cisternae studied, there are 394 vesicles (8 clathrin coated, 190 nonclathrin coated, and 196 noncoated), indicating considerable vesicular traffic in this Golgi region. Our data place structural constraints on models of trafficking to, through, and from the Golgi complex.


Subject(s)
Golgi Apparatus/ultrastructure , Image Processing, Computer-Assisted/methods , Kidney/ultrastructure , Animals , Biological Transport, Active , Cells, Cultured , Computer Graphics , Computer Simulation , Cryoelectron Microscopy , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/ultrastructure , Freeze Substitution , Golgi Apparatus/metabolism , Kidney/metabolism , Lipid Metabolism , Models, Anatomic , Models, Biological , Rats
10.
J Biol Chem ; 273(17): 10349-54, 1998 Apr 24.
Article in English | MEDLINE | ID: mdl-9553090

ABSTRACT

Current evidence suggests that phosphatidylinositol (PI) kinases and phosphatidylinositol transfer protein (PITP) are involved in driving vesicular traffic from yeast and mammalian trans-Golgi network (TGN). We have tested the interaction between these cytosolic proteins in an assay that measures the formation of constitutive transport vesicles from the TGN in a hepatocyte cell-free system. This reaction is dependent on a novel PI 3-kinase, and we now report that, under conditions of limiting cytosol, purified PI 3-kinase and PITP functionally cooperate to drive exocytic vesicle formation. This synergy was observed with both yeast and mammalian PITPs, and it also extended to the formation of PI 3-phosphate. These collective findings indicate that the PI 3-kinase and PITP synergize to form a pool of PI 3-phosphate that is essential for formation of exocytic vesicles from the hepatocyte TGN.


Subject(s)
Carrier Proteins/metabolism , Golgi Apparatus/metabolism , Membrane Proteins , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylinositols/metabolism , Saccharomyces cerevisiae Proteins , Animals , Biological Transport , Exocytosis , Liver/metabolism , Liver/ultrastructure , Phospholipid Transfer Proteins , Rats , Substrate Specificity
11.
Science ; 279(5350): 573-7, 1998 Jan 23.
Article in English | MEDLINE | ID: mdl-9438853

ABSTRACT

Dynamin guanosine triphosphatases support the scission of clathrin-coated vesicles from the plasmalemma during endocytosis. By fluorescence microscopy of cultured rat hepatocytes, a green fluorescent protein-dynamin II fusion protein localized with clathrin-coated vesicles at the Golgi complex. A cell-free assay was utilized to demonstrate the role of dynamin in vesicle formation at the trans-Golgi. Addition of peptide-specific anti-dynamin antibodies to the assay mixture inhibited both constitutive exocytic and clathrin-coated vesicle formation. Immunodepletion of dynamin proteins also inhibited vesicle formation, and budding efficiency was restored upon readdition of purified dynamin. These data suggest that dynamin participates in the formation of distinct transport vesicles from the trans-Golgi network.


Subject(s)
Coated Vesicles/metabolism , GTP Phosphohydrolases/metabolism , Golgi Apparatus/metabolism , Organelles/metabolism , Adenosine Triphosphate/metabolism , Animals , Biological Transport , Cell Membrane/chemistry , Cells, Cultured , Clathrin/analysis , Coated Vesicles/chemistry , Coated Vesicles/ultrastructure , Cytosol/metabolism , Dynamins , GTP Phosphohydrolases/analysis , GTP Phosphohydrolases/immunology , Golgi Apparatus/chemistry , Green Fluorescent Proteins , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Liver/ultrastructure , Luminescent Proteins , Microscopy, Fluorescence , Organelles/chemistry , Organelles/ultrastructure , Rats , Recombinant Fusion Proteins/analysis
12.
Mol Biol Cell ; 8(10): 1911-31, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9348533

ABSTRACT

To characterize endogenous molecules and activities of the Golgi complex, proteins in transit were > 99% cleared from rat hepatocytes by using cycloheximide (CHX) treatment. The loss of proteins in transit resulted in condensation of the Golgi cisternae and stacks. Isolation of a stacked Golgi fraction is equally efficient with or without proteins in transit [control (CTL SGF1) and cycloheximide (CHX SGF1)]. Electron microscopy and morphometric analysis showed that > 90% of the elements could be positively identified as Golgi stacks or cisternae. Biochemical analysis showed that the cis-, medial-, trans-, and TGN Golgi markers were enriched over the postnuclear supernatant 200- to 400-fold with and 400- to 700-fold without proteins in transit. To provide information on a mechanism for import of calcium required at the later stages of the secretory pathway, calcium uptake into CTL SGF1 and CHX SGF1 was examined. All calcium uptake into CTL SGF1 was dependent on a thapsigargin-resistant pump not resident to the Golgi complex and a thapsigargin-sensitive pump resident to the Golgi. Experiments using CHX SGF1 showed that the thapsigargin-resistant activity was a plasma membrane calcium ATPase isoform in transit to the plasma membrane and the thapsigargin-sensitive pump was a sarcoplasmic/endoplasmic reticulum calcium ATPase isoform. In vivo both of these calcium ATPases function to maintain millimolar levels of calcium within the Golgi lumen.


Subject(s)
Calcium/pharmacokinetics , Golgi Apparatus/metabolism , Golgi Apparatus/ultrastructure , Proteins/metabolism , Adenosine Triphosphatases/antagonists & inhibitors , Adenosine Triphosphate/pharmacology , Animals , Biomarkers/analysis , Calcium/antagonists & inhibitors , Calcium-Transporting ATPases/metabolism , Cell Compartmentation , Cell Membrane/enzymology , Cell Membrane/ultrastructure , Cycloheximide/pharmacology , Data Interpretation, Statistical , Endoplasmic Reticulum/chemistry , Endoplasmic Reticulum/ultrastructure , Enzyme Inhibitors/pharmacology , Golgi Apparatus/drug effects , Immunoblotting , Intracellular Membranes/drug effects , Intracellular Membranes/metabolism , Intracellular Membranes/ultrastructure , Proteins/drug effects , Rats , Sarcoplasmic Reticulum/enzymology , Tissue Distribution
13.
J Cell Biol ; 139(2): 339-49, 1997 Oct 20.
Article in English | MEDLINE | ID: mdl-9334339

ABSTRACT

An 85-kD cytosolic complex (p62(cplx)), consisting of a 62-kD phosphoprotein (p62) and a 25-kD GTPase, has been shown to be essential for the cell-free reconstitution of polymeric IgA receptor (pIgA-R)-containing exocytic transport vesicle formation from the TGN (Jones, S.M., J.R. Crosby, J. Salamero, and K.E. Howell. 1993. J. Cell Biol. 122:775-788). Here the p62(cplx) is identified as a regulatory subunit of a novel phosphatidylinositol 3-kinase (PI3-kinase). This p62(cplx)-associated PI3-kinase activity is stimulated by activation of the p62(cplx)-associated GTPase, and is specific for phosphatidylinositol (PI) as substrate, and is sensitive to wortmannin at micromolar concentrations. The direct role of this p62(cplx)-associated PI3-kinase activity in TGN-derived vesicle formation is indicated by the finding that both lipid kinase activity and the formation of pIgA-R-containing exocytic vesicles from the TGN are inhibited by wortmannin with similar dose-response curves and 50% inhibitory concentrations (3.5 microM). These findings indicate that phosphatidylinositol-3-phosphate (PI[3]P) is required for the formation of TGN-derived exocytic transport vesicles, and that the p62(cplx)-associated PI3-kinase and an activated GTPase are the essential molecules that drive production of this PI(3)P.


Subject(s)
Golgi Apparatus/metabolism , Liver/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Receptors, Fc/biosynthesis , Amino Acid Sequence , Androstadienes/pharmacology , Animals , Cattle , Cytoplasmic Granules/metabolism , Cytosol/metabolism , Enzyme Inhibitors/pharmacology , Exocytosis , GTP Phosphohydrolases/metabolism , Golgi Apparatus/immunology , Humans , Immunoglobulin A/metabolism , Kinetics , Macromolecular Substances , Mice , Molecular Sequence Data , Phosphatidylinositol 3-Kinases/chemistry , Phosphoproteins/metabolism , Rats , Receptors, Fc/chemistry , Sequence Alignment , Sequence Homology, Amino Acid , Wortmannin
14.
Electrophoresis ; 18(14): 2601-12, 1997 Dec.
Article in English | MEDLINE | ID: mdl-9527490

ABSTRACT

The discovery of additional endogenous Golgi proteins will lead to significant new insights into Golgi function. To this end, stacked Golgi fractions (SGFs) were isolated from rat liver before (CTL SGF) and after molecules in transit through the Golgi were cleared by pre-treatment with cycloheximide (CHX SGF). Electron microscopic (EM) morphometric and biochemical analyses showed that the in vivo stacked morphology is retained, that > 90% of the elements can be positively identified as Golgi stacks and cisternae, and that transmembrane protein markers of the Golgi complex are enriched 300- to 800-fold over starting postnuclear supernatant (PNS). High-resolution two-dimensional (2-D) gel mapping has been carried out on the CTL PNS, CTL SII (an intermediate fraction), CTL SGF, CHX SGF, CHX SGF - high pH supernatant, and CHX SGF - high pH pellet. This analysis, coupled with immunoblotting and alignment of the 2-D gels with master gels, has allowed the identification of a number of known proteins and the preliminary characterization of the most abundant 173 Golgi-specific proteins. These 173 proteins have been placed into three categories: cargo, cytosolic Golgi-associated, and resident Golgi proteins. These categories are tentative and will be modified as more data are acquired from immunoblotting and protein sequencing.


Subject(s)
Electrophoresis, Gel, Two-Dimensional/methods , Golgi Apparatus/chemistry , Peptide Mapping/methods , Proteins/analysis , Animals , Cytosol/chemistry , Liver/chemistry , Rats
15.
Mol Cell Biol ; 16(7): 3275-84, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8668142

ABSTRACT

Vesicle-mediated traffic between compartments of the yeast secretory pathway involves recruitment of multiple cytosolic proteins for budding, targeting, and membrane fusion events. The SEC7 gene product (Sec7p) is a constituent of coat structures on transport vesicles en route to the Golgi complex in the yeast Saccharomyces cerevisiae. To identify mammalian homologs of Sec7p and its interacting proteins, we used a genetic selection strategy in which a human HepG2 cDNA library was transformed into conditional-lethal yeast sec7 mutants. We isolated several clones capable of rescuing sec7 mutant growth at the restrictive temperature. The cDNA encoding the most effective suppressor was identified as human ADP ribosylation factor 4 (hARF4), a member of the GTPase family proposed to regulate recruitment of vesicle coat proteins in mammalian cells. Having identified a Sec7p-interacting protein rather than the mammalian Sec7p homolog, we provide evidence that hARF4 suppressed the sec7 mutation by restoring secretory pathway function. Shifting sec7 strains to the restrictive temperature results in the disappearance of the mutant Sec7p cytosolic pool without apparent changes in the membrane-associated fraction. The introduction of hARF4 to the cells maintained the balance between cytosolic and membrane-associated Sec7p pools. These results suggest a requirement for Sec7p cycling on and off of the membranes for cell growth and vesicular traffic. In addition, overexpression of the yeast GTPase-encoding genes ARF1 and ARF2, but not that of YPT1, suppressed the sec7 mutant growth phenotype in an allele-specific manner. This allele specificity indicates that individual ARFs are recruited to perform two different Sec7p-related functions in vesicle coat dynamics.


Subject(s)
Fungal Proteins/genetics , GTP-Binding Proteins/biosynthesis , Guanine Nucleotide Exchange Factors , Saccharomyces cerevisiae/growth & development , ADP-Ribosylation Factor 1 , ADP-Ribosylation Factors , Carrier Proteins/biosynthesis , Cloning, Molecular , DNA, Complementary , Enzyme Induction , Fungal Proteins/biosynthesis , Fungal Proteins/metabolism , GTP-Binding Proteins/metabolism , Gene Library , Genetic Complementation Test , Glycoside Hydrolases/biosynthesis , Humans , Kinetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Suppression, Genetic , Tumor Cells, Cultured , beta-Fructofuranosidase
17.
J Cell Biol ; 127(1): 29-38, 1994 Oct.
Article in English | MEDLINE | ID: mdl-7929568

ABSTRACT

High voltage electron microscopy and computer axial tomography have been used to study the 3-D structure of trans-Golgi cisternae and trans-Golgi networks (TGNs) in NRK cells. Both structures were specifically labeled by photoconversion of a fluorescent analogue of ceramide using a modification of the techique of Pagano et al. (J. Cell Biol. 1991. 113: 1267-1279). Regions of the Golgi ribbon in fixed, stained cells were cut in 250-nm sections and analyzed by tilt series microscopy and subsequent tomographic reconstruction. Resolution of the reconstructions ranged from 6 to 10 nm. The size and structure of the TGN varied considerably throughout the Golgi ribbon; all reconstructions were made from regions with pronounced TGN. Most regions analyzed contained multiple (2-4) Golgi cisternae that stain with ceramide. These "peel off" from the closely stacked cisternae and are continuous at their ends with tubules that contribute to the TGN. Most vesicular profiles visualized in the TGN are connected to TGN tubules. The budding of vesicles appears to occur synchronously along the length of a TGN tubule. Two distinct coats were visualized on budding vesicles: clathrin cages and a novel, lace-like structure. Individual TGN tubules produce vesicles of only one coat type. These observations lead to the following predictions: (a) sorting of molecules must occur prior to the formation of TGN tubules; (b) vesicle formation takes place almost synchronously along a given TGN tubule; and (c) lace-like coats form an exocytic vesicles.


Subject(s)
Golgi Apparatus/ultrastructure , Microscopy, Electron/methods , Animals , Cell Line , Ceramides , Coated Pits, Cell-Membrane/ultrastructure , Fluorescent Dyes , Tomography/methods
18.
Trends Cell Biol ; 3(8): 252-5, 1993 Aug.
Article in English | MEDLINE | ID: mdl-14731742

ABSTRACT

TGN38/41 is a heterodimeric integral membrane protein that cycles between the trans Golgi network and the cell surface. A tyrosine-containing tetrapeptide motif within its cytoplasmic tail is necessary and sufficient for determining its steady-state location in the TGN. Recent results have shown that TGN38/41 plays an essential role in the formation of exocytic vesicles at the TGN by serving as a receptor for complexes of a cytoplasmic protein known as p62, and one of four small GTP-binding proteins, including rab6. For budding to occur, this complex must bind to the cytoplasmic domain of TGN38/41. We propose here that TGN38/41 may couple the segregation of secretory proteins to the budding of exocytic vesicles at the TGN.

19.
J Cell Biol ; 122(4): 775-88, 1993 Aug.
Article in English | MEDLINE | ID: mdl-8349729

ABSTRACT

TGN38/41, an integral membrane protein predominantly localized to the trans-Golgi network, has been shown to cycle to the plasma membrane and return to the TGN within 30 min. (Ladinsky, M. S., and K. E. Howell. 1992. Eur. J. Cell Biol. 59:92-105). In characterizing the proteins which associate with TGN38/41, a peripheral 62-kD protein, two forms of rab6 and two other small GTP-binding proteins were identified by coimmunoprecipitation. However, approximately 90% of the 62-kD protein is cytosolic and is associated with the same subset of small GTP-binding proteins. Both the membrane and cytoplasmic complexes were characterized by sizing column fractionation and velocity sedimentation. The membrane complex was approximately 250 kD (11.6 S) consisting of the cytosolic complex and a heterodimer of TGN38/41 (160 kD). The cytosolic complex was approximately 86 kD (6.1 S) consisting of p62 and one small GTP-binding protein. Preliminary evidence indicates that phosphorylation of the p62 molecule regulates the dissociation of the cytosolic complex from TGN38/41. Functionally the cytosolic p62 complex must bind to TGN38/41 for the budding of exocytic transport vesicles from the TGN as assayed in a cell-free system (Salamero, J., E. S. Sztul, and K. E. Howell. 1990. Proc. Natl. Acad. Sci. USA. 87:7717-7721). Interference with p62, rab6 or TGN38, and TGN41 cytoplasmic domains by immunodepletion or competing peptides completely inhibited the budding of exocytic transport vesicles. These results support an essential role for interaction of the cytosolic p62/rab6 complex with TGN38/41 in budding of exocytic vesicles from the TGN.


Subject(s)
Exocytosis , Glycoproteins , Golgi Apparatus/metabolism , Membrane Glycoproteins/metabolism , Membrane Proteins , Amino Acid Sequence , Animals , Biological Transport , Cell Compartmentation , Cell Line , Cell Membrane/metabolism , Cytosol/metabolism , GTP-Binding Proteins/metabolism , Golgi Apparatus/chemistry , In Vitro Techniques , Intracellular Membranes/metabolism , Macromolecular Substances , Molecular Sequence Data , Peptides/chemistry , Phosphoproteins/metabolism , Phosphorylation , Precipitin Tests , Rats
20.
J Cell Sci Suppl ; 17: 41-7, 1993.
Article in English | MEDLINE | ID: mdl-8144704

ABSTRACT

We have used electron microscopy to further characterize details of the dynamics of TGN38/41, a protein found to cycle between the trans-Golgi network and the plasma membrane. Immunogold-labeling of NRK cells under steady-state conditions shows the majority of TGN38/41 is localized to the trans-most Golgi cisternae and the trans-Golgi network. Small amounts of this molecule can be detected in early endosomes. Capture of cycling TGN38/41 molecules at the cell surface altered the steady state distribution. This was accomplished by binding TGN38/41 luminal domain antibodies to solid supports (beads), which were introduced to the culture media of cells. As increasing numbers of antigen-antibody complexes formed, the beads were internalized by the 'zippering mechanism' of phagocytosis. This provides a system that can address many questions related to the function of TGN38/41 and the trans-Golgi network itself.


Subject(s)
Cell Polarity/physiology , Glycoproteins , Golgi Apparatus/metabolism , Golgi Apparatus/ultrastructure , Membrane Glycoproteins/metabolism , Membrane Proteins , Animals , Biological Transport, Active , Cell Line , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Endocytosis , Kidney/metabolism , Kidney/ultrastructure , Microscopy, Immunoelectron , Rats
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