Immunoisolation of Kex2p-containing organelles from yeast demonstrates colocalisation of three processing proteinases to a single Golgi compartment

1993 ◽  
Vol 106 (3) ◽  
pp. 815-822
Author(s):  
N.J. Bryant ◽  
A. Boyd
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Secretory Pathway ◽  
High Yield ◽  
Trans Golgi Network ◽  
Processing Enzymes ◽  
Terminal Domain ◽  
Golgi Compartment ◽  
Functional Equivalent ◽  
Golgi Network

One of the Golgi compartments of Saccharomyces cerevisiae is defined by the presence of a specific endoproteinase, Kex2p, which cleaves precursor polypeptides at pairs of basic residues. We have used antibodies directed against the cytoplasmically disposed C-terminal domain of Kex2p to develop an immuno-affinity procedure for the isolation of Kex2p-containing organelles. The method gives a high yield of sealed organelles that are essentially free of contamination from other secretory pathway organelles while being significantly enriched for two other late Golgi enzymes, dipeptidylaminopeptidase A and the Kex1 carboxypeptidase. Our findings provide clear evidence for a single yeast Golgi compartment containing all three late-processing enzymes, which is likely to be the functional equivalent in yeast of the mammalian trans-Golgi network.

Differential effects of temperature blockade on the proteolytic processing of three secretory granule-associated proteins

1994 ◽  
Vol 107 (3) ◽  
pp. 737-745 ◽  
Author(s):  
S.L. Milgram ◽  
R.E. Mains
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Proteolytic Processing ◽  
Prohormone Convertase ◽  
Trans Golgi Network ◽  
Processing Enzymes ◽  
Prohormone Convertase 1 ◽  
Associated Proteins ◽  
Mouse Pituitary ◽  
Golgi Network

Vesicular transport within the secretory pathway can be arrested by incubating cells at 15 degrees C or 20 degrees C to block exit from the endoplasmic reticulum or trans-Golgi network, respectively. Using this powerful tool we have compared the intracellular sites of endoproteolytic processing of proopiomelanocortin and two prohormone processing enzymes in AtT-20 mouse pituitary corticotrope tumor cells. For comparison, proopiomelanocortin processing was also evaluated in primary neurointermediate pituitary cultures. AtT-20 cells synthesize and store endogenous proopiomelanocortin and prohormone convertase 1; AtT-20 cells expressing high levels of integral membrane or soluble peptidylglycine alpha-amidating monooxygenase were generated by stable transfection. Cells were incubated with [35S]methionine and chased at 4 degrees C, 15 degrees C, 20 degrees C or 37 degrees C. The endoproteolytic processing of peptidylglycine alpha-amidating mono-oxygenase, prohormone convertase 1, and proopiomelanocortin was compared following immunoprecipitation. Endoproteolytic processing of integral membrane and soluble peptidylglycine alpha-amidating monooxygenase proteins was completely blocked by incubation of cells at 20 degrees C. In contrast, prohormone convertase 1 processing from the 87 kDa precursor to the 81 kDa intermediate proceeded to completion at both 15 degrees C and 20 degrees C, while cleavage to generate the 63 kDa prohormone convertase 1 protein was completely blocked at 20 degrees C. In AtT-20 cells and neurointermediate pituitary cultures, generation of beta-lipotropin from proopiomelanocortin continued at a slow but significant rate at 20 degrees C, while processing of beta-lipotropin to beta-endorphin was blocked. Thus prohormone convertase 1 processing begins in the endoplasmic reticulum and is not completed until after the trans-Golgi network, while peptidylglycine alpha-amidating monooxygenase processing begins after the trans-Golgi network. Selected proopiomelanocortin cleavages begin before entry into immature granules.

scholarly journals Ionic milieu controls the compartment-specific activation of pro-opiomelanocortin processing in AtT-20 cells.

1995 ◽  
Vol 6 (10) ◽  
pp. 1271-1285 ◽  
Author(s):  
W K Schmidt ◽  
H P Moore
Keyword(s):  
Intracellular Transport ◽  
Secretory Pathway ◽  
Intact Cell ◽  
Secretory Granules ◽  
Cell System ◽  
Proteolytic Processing ◽  
Prolonged Incubation ◽  
Trans Golgi Network ◽  
Processing Enzymes ◽  
Golgi Network

Newly synthesized prohormones and their processing enzymes transit through the same compartments before being packaged into regulated secretory granules. Despite this coordinated intracellular transport, prohormone processing does not occur until late in the secretory pathway. In the mouse pituitary AtT-20 cell line, conversion of pro-opiomelanocortin (POMC) to mature adrenocorticotropic hormone involves the prohormone convertase PC1. The mechanism by which this proteolytic processing is restricted to late secretory compartments is unknown; PC1 activity could be regulated by compartment-specific activators/inhibitors, or through changes in the ionic milieu that influence its activity. By arresting transport in a semi-intact cell system, we have addressed whether metabolically labeled POMC trapped in early secretory compartments can be induced to undergo conversion if the ionic milieu in these compartments is experimentally manipulated. Prolonged incubation of labeled POMC trapped in the endoplasmic reticulum or Golgi/trans-Golgi network did not result in processing, thereby supporting the theory that processing is normally a post-Golgi/trans-Golgi network event. However, acidification of these compartments allowed effective processing of POMC to the intermediate and mature forms. The observed processing increased sharply at a pH below 6.0 and required millimolar calcium, regardless of the compartment in which labeled POMC resided. These conditions also resulted in the coordinate conversion of PC1 from the 84/87 kDa into the 74-kDa and 66-kDa forms. We propose that POMC processing is predominantly restricted to acidifying secretory granules, and that a change in pH within these granules is both necessary and sufficient to activate POMC processing.

The Alzheimer's Amyloid Precursor Is Cleaved Intracellularly in the Trans-Golgi Network or in a Post-Golgi Compartment

1992 ◽  
Vol 674 (1 Proteases and) ◽  
pp. 118-128 ◽  
Author(s):  
K. SAMBAMURTI ◽  
L. M. REFOLO ◽  
J. SHIOI ◽  
M. A. PAPPOLLA ◽  
N. K. ROBAKIS
Keyword(s):  
Trans Golgi Network ◽  
Golgi Compartment ◽  
Golgi Network

scholarly journals Cofilin-mediated sorting and export of specific cargo from the Golgi apparatus in yeast

2012 ◽  
Vol 23 (12) ◽  
pp. 2327-2338 ◽  
Author(s):  
Amy J. Curwin ◽  
Julia von Blume ◽  
Vivek Malhotra
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
Calcium Atpase ◽  
Carboxypeptidase Y ◽  
Secretory Proteins ◽  
Golgi Membranes ◽  
Golgi Compartment ◽  
Reduced Rate ◽  
Golgi Network

The mechanism of cargo sorting at the trans-Golgi network (TGN) for secretion is poorly understood. We previously reported the involvement of the actin-severing protein cofilin and the Ca2+ ATPase secretory pathway calcium ATPase 1 (SPCA1) in the sorting of soluble secretory cargo at the TGN in mammalian cells. Now we report that cofilin in yeast is required for export of selective secretory cargo at the late Golgi membranes. In cofilin mutant (cof1-8) cells, the cell wall protein Bgl2 was secreted at a reduced rate and retained in a late Golgi compartment, whereas the plasma membrane H+ ATPase Pma1, which is transported in the same class of carriers, reached the cell surface. In addition, sorting of carboxypeptidase Y (CPY) to the vacuole was delayed, and CPY was secreted from cof1-8 cells. Loss of the yeast orthologue of SPCA1 (Pmr1) exhibited similar sorting defects and displayed synthetic sickness with cof1-8. In addition, overexpression of PMR1 restored Bgl2 secretion in cof1-8 cells. These findings highlight the conserved role of cofilin and SPCA1/Pmr1 in sorting of the soluble secretory proteins at the TGN/late Golgi membranes in eukaryotes.

scholarly journals Intracellular movement of two mannose 6-phosphate receptors: return to the Golgi apparatus.

1988 ◽  
Vol 106 (3) ◽  
pp. 617-628 ◽  
Author(s):  
J R Duncan ◽  
S Kornfeld
Keyword(s):  
Sialic Acid ◽  
Golgi Apparatus ◽  
Chinese Hamster ◽  
Murine Lymphoma ◽  
Trans Golgi Network ◽  
Intracellular Movement ◽  
Golgi Region ◽  
Golgi Compartment ◽  
Mannose 6 Phosphate ◽  
Golgi Network

We have used Chinese hamster ovary (CHO) cells and a murine lymphoma cell line to study the recycling of the 215-kD and the 46-kD mannose 6-phosphate receptors to various regions of the Golgi to determine the site where the receptors first encounter newly synthesized lysosomal enzymes. For assessing return to the trans-most Golgi compartments containing sialyltransferase (trans-cisternae and trans-Golgi network), the oligosaccharides of receptor molecules on the cell surface were labeled with [3H]galactose at 4 degrees C. Upon warming to 37 degrees C, the [3H]galactose residues on both receptors were substituted with sialic acid with a t1/2 approximately 3 hrs. Other glycoproteins acquired sialic acid at least 8-10 times slower. Return of the receptors to the trans-Golgi cisternae containing galactosyltransferase could not be detected. Return to the cis/middle Golgi cisternae containing alpha-mannosidase I was measured by adding deoxymannojirimycin, a mannosidase I inhibitor, during the initial posttranslational passage of [3H]mannose-labeled glycoproteins through the Golgi, thereby preserving oligosaccharides which would be substrates for alpha-mannosidase I. After removal of the inhibitor, return to the early Golgi with subsequent passage through the Golgi complex was measured by determining the conversion of the oligosaccharides from high mannose to complex-type units. This conversion was very slow for the receptors and other glycoproteins (t1/2 approximately 20 h). Exposure of the receptors and other glycoproteins to the dMM-sensitive alpha-mannosidase without movement through the Golgi apparatus was determined by measuring the loss of mannose residues from these proteins. This loss was also slow. These results indicate that both Man-6-P receptors routinely return to the Golgi compartment which contains sialyltransferase and recycle through other regions of the Golgi region less frequently. We infer that the trans-Golgi network is the major site for lysosomal enzyme sorting in CHO and murine lymphoma cells.

scholarly journals Ent3p and Ent5p Exhibit Cargo-specific Functions in Trafficking Proteins between the Trans-Golgi Network and the Endosomes in Yeast

2007 ◽  
Vol 18 (5) ◽  
pp. 1803-1815 ◽  
Author(s):  
Alenka Čopič ◽  
Trevor L. Starr ◽  
Randy Schekman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Surface ◽  
Protein Transport ◽  
Adaptor Protein ◽  
Additional Function ◽  
Trans Golgi Network ◽  
Cargo Proteins ◽  
Clathrin Adaptor ◽  
Dependent Transport ◽  
Golgi Network

The phosphoinositide-binding proteins Ent3p and Ent5p are required for protein transport from the trans-Golgi network (TGN) to the vacuole in Saccharomyces cerevisiae. Both proteins interact with the monomeric clathrin adaptor Gga2p, but Ent5p also interacts with the clathrin adaptor protein 1 (AP-1) complex, which facilitates retention of proteins such as Chs3p at the TGN. When both ENT3 and ENT5 are mutated, Chs3p is diverted from an intracellular reservoir to the cell surface. However, Ent3p and Ent5p are not required for the function of AP-1, but rather they seem to act in parallel with AP-1 to retain proteins such as Chs3p at the TGN. They have all the properties of clathrin adaptors, because they can both bind to clathrin and to cargo proteins. Like AP-1, Ent5p binds to Chs3p, whereas Ent3p facilitates the interaction between Gga2p and the endosomal syntaxin Pep12p. Thus, Ent3p has an additional function in Gga-dependent transport to the late endosome. Ent3p also facilitates the association between Gga2p and clathrin; however, Ent5p can partially substitute for this function. We conclude that the clathrin adaptors AP-1, Ent3p, Ent5p, and the Ggas cooperate in different ways to sort proteins between the TGN and the endosomes.

scholarly journals Proteomic Characterization of Isolated Arabidopsis Clathrin-Coated Vesicles Reveals Evolutionarily Conserved and Plant Specific Components

2021 ◽  
Author(s):  
Dana A. Dahhan ◽  
Gregory D. Reynolds ◽  
Jessica J. Cárdenas ◽  
Dominique Eeckhout ◽  
Alexander Johnson ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Adaptor Protein ◽  
Coated Vesicles ◽  
Mass Spectrometry Analysis ◽  
Chemical Labeling ◽  
Spectrometry Analysis ◽  
Trans Golgi Network ◽  
Evolutionarily Conserved ◽  
Golgi Network

In eukaryotes, clathrin-coated vesicles (CCVs) facilitate the internalization of material from the cell surface as well as the movement of cargo in post-Golgi trafficking pathways. This diversity of functions is partially provided by multiple monomeric and multimeric clathrin adaptor complexes that provide compartment and cargo selectivity. The adaptor-protein AP-1 complex operates as part of the secretory pathway at the trans-Golgi network, while the AP-2 complex and the TPLATE complex (TPC) jointly operate at the plasma membrane to execute clathrin-mediated endocytosis. Key to our further understanding of clathrin-mediated trafficking in plants will be the comprehensive identification and characterization of the network of evolutionarily conserved and plant-specific core and accessory machinery involved in the formation and targeting of CCVs. To facilitate these studies, we have analyzed the proteome of enriched trans-Golgi network/early endosome-derived and endocytic CCVs isolated from dividing and expanding suspension-cultured Arabidopsis cells. Tandem mass spectrometry analysis results were validated by differential chemical labeling experiments to identify proteins co-enriching with CCVs. Proteins enriched in CCVs included previously characterized CCV components and cargos such as the vacuolar sorting receptors in addition to conserved and plant-specific components whose function in clathrin-mediated trafficking has not been previously defined. Notably, in addition to AP-1 and AP-2, all subunits of the AP-4 complex, but not AP-3 or AP-5, were found to be in high abundance in the CCV proteome. The association of AP-4 with suspension-cultured Arabidopsis CCVs is further supported via additional biochemical data.

Immunolocalization of the intracellular sites of accumulation of mutant influenza hemagglutinins by Electron Microscopy

1989 ◽  
Vol 47 ◽  
pp. 968-969
Author(s):  
K. McCammon ◽  
M. Segal ◽  
J. Sambrook ◽  
M. J. Gething ◽  
A. McDowall
Keyword(s):  
Electron Microscopy ◽  
Golgi Apparatus ◽  
Mammalian Cells ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Cysteine Residue ◽  
Homologous Sequence ◽  
Golgi Compartment ◽  
Golgi Network

The hemagglutinin (HA) of influenza virus has been used as a model system to study the biosynthesis and intracellular transport of integral membrane proteins in mammalian cells. To investigate the role of protein structure in facilitating transport along the secretory pathway, we have examined the expression in monkey CV-1 cells of a large number of mutant HA molecules. The majority of the HA mutants do not progress along the secretory pathway and accumulate in the endoplasmic reticulum (ER), and we have shown that assembly of newly-synthesized HA monomers into correctly folded trimeric structures is required for transport of the protein to the Golgi apparatus. By contrast, only one HA mutant has beegn characterized whose transport is blocked at a post-Golgi stage of the pathway and thus little is known about the factors involved in the sorting of the HA molecule from the Golgi apparatus to the plasma membrane (PM). In this study we are using electron microscopy to precisely define the intracellular site of accumulation of two mutant HAs whose transport is blocked at different stages of the secretory pathway. In mutant HAJS67, a cysteine residue (cys67) involved in a key disulfide bond has been substituted by a serine residue. In mutant HA164, the 10 amino acid cytoplasmic tail of the wild-type HA has been replaced by a non-homologous sequence of 16 amino acids. Biochemical and immunof1uoresence analyses have indicated that HAJS67 molecules remain in the ER compartment while HA164 is largely confined to a post-Golgi compartment, possibly the trans Golgi network (TGN).

scholarly journals Immunolocalization of Kex2 protease identifies a putative late Golgi compartment in the yeast Saccharomyces cerevisiae.

1991 ◽  
Vol 113 (3) ◽  
pp. 527-538 ◽  
Author(s):  
K Redding ◽  
C Holcomb ◽  
R S Fuller
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Secretory Pathway ◽  
Killer Toxin ◽  
Proteolytic Processing ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Golgi Compartment ◽  
Membrane Bound ◽  
Gene Structures ◽  
Stage Analysis

The Kex2 protein of the yeast Saccharomyces cerevisiae is a membrane-bound, Ca2(+)-dependent serine protease that cleaves the precursors of the mating pheromone alpha-factor and the M1 killer toxin at pairs of basic residues during their transport through the secretory pathway. To begin to characterize the intracellular locus of Kex2-dependent proteolytic processing, we have examined the subcellular distribution of Kex2 protein in yeast by indirect immunofluorescence. Kex2 protein is located at multiple, discrete sites within wild-type yeast cells (average, 3.0 +/- 1.7/mother cell). Qualitatively similar fluorescence patterns are observed at elevated levels of expression, but no signal is found in cells lacking the KEX2 gene. Structures containing Kex2 protein are not concentrated at a perinuclear location, but are distributed throughout the cytoplasm at all phases of the cell cycle. Kex2-containing structures appear in the bud at an early, premitotic stage. Analysis of conditional secretory (sec) mutants demonstrates that Kex2 protein ordinarily progresses from the ER to the Golgi but is not incorporated into secretory vesicles, consistent with the proposed localization of Kex2 protein to the yeast Golgi complex.

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