scholarly journals Visualization of secretory cargo transport within the Golgi apparatus

2019 ◽  
Vol 218 (5) ◽  
pp. 1602-1618 ◽  
Author(s):  
Kazuo Kurokawa ◽  
Hiroko Osakada ◽  
Tomoko Kojidani ◽  
Miho Waga ◽  
Yasuyuki Suda ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Golgi Apparatus ◽  
Yeast Saccharomyces Cerevisiae ◽  
Trans Golgi Network ◽  
Golgi Cisterna ◽  
Cargo Transport ◽  
Golgi Network ◽  
Interesting Behavior

To describe trafficking of secretory cargo within the Golgi apparatus, the cisternal maturation model predicts that Golgi cisternae change their properties from cis to trans while cargo remains in the cisternae. Cisternal change has been demonstrated in living yeast Saccharomyces cerevisiae; however, the behavior of cargo has yet to be examined directly. In this study, we conducted simultaneous three-color and four-dimensional visualization of secretory transmembrane cargo together with early and late Golgi resident proteins. We show that cargo stays in a Golgi cisterna during maturation from cis-Golgi to trans-Golgi and further to the trans-Golgi network (TGN), which involves dynamic mixing and segregation of two zones of the earlier and later Golgi resident proteins. The location of cargo changes from the early to the late zone within the cisterna during the progression of maturation. In addition, cargo shows an interesting behavior during the maturation to the TGN. After most cargo has reached the TGN zone, a small amount of cargo frequently reappears in the earlier zone.

Identification of a di-leucine motif within the C terminus domain of the Menkes disease protein that mediates endocytosis from the plasma membrane

1999 ◽  
Vol 112 (11) ◽  
pp. 1721-1732 ◽  
Author(s):  
M.J. Francis ◽  
E.E. Jones ◽  
E.R. Levy ◽  
R.L. Martin ◽  
S. Ponnambalam ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Transmembrane Domain ◽  
Menkes Disease ◽  
Cytoplasmic Domain ◽  
Endocytic Pathway ◽  
Site Directed Mutagenesis ◽  
Trans Golgi Network ◽  
C Terminus ◽  
Golgi Network

The protein encoded by the Menkes disease gene (MNK) is localised to the Golgi apparatus and cycles between the trans-Golgi network and the plasma membrane in cultured cells on addition and removal of copper to the growth medium. This suggests that MNK protein contains active signals that are involved in the retention of the protein to the trans-Golgi network and retrieval of the protein from the plasma membrane. Previous studies have identified a signal involved in Golgi retention within transmembrane domain 3 of MNK. To identify a motif sufficient for retrieval of MNK from the plasma membrane, we analysed the cytoplasmic domain, downstream of transmembrane domain 7 and 8. Chimeric constructs containing this cytoplasmic domain fused to the reporter molecule CD8 localised the retrieval signal(s) to 62 amino acids at the C terminus. Further studies were performed on putative internalisation motifs, using site-directed mutagenesis, protein expression, chemical treatment and immunofluorescence. We observed that a di-leucine motif (L1487L1488) was essential for rapid internalisation of chimeric CD8 proteins and the full-length Menkes cDNA from the plasma membrane. We suggest that this motif mediates the retrieval of MNK from the plasma membrane into the endocytic pathway, via the recycling endosomes, but is not sufficient on its own to return the protein to the Golgi apparatus. These studies provide a basis with which to identify other motifs important in the sorting and delivery of MNK from the plasma membrane to the Golgi apparatus.

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.

scholarly journals Transition of Galactosyltransferase 1 from Trans-Golgi Cisterna to the Trans-Golgi Network Is Signal Mediated

2006 ◽  
Vol 17 (12) ◽  
pp. 5153-5162 ◽  
Author(s):  
Beat E. Schaub ◽  
Bea Berger ◽  
Eric G. Berger ◽  
Jack Rohrer
Keyword(s):  
Amino Acids ◽  
Cytoplasmic Tail ◽  
Time Lapse ◽  
Trans Golgi Network ◽  
Golgi Cisterna ◽  
Confocal Fluorescence ◽  
Rapid Accumulation ◽  
Gradient Fractionation ◽  
Golgi Network ◽  
Complex Glycan

The Golgi apparatus (GA) is the organelle where complex glycan formation takes place. In addition, it is a major sorting site for proteins destined for various subcellular compartments or for secretion. Here we investigate β1,4-galactosyltransferase 1 (galT) and α2,6-sialyltransferase 1 (siaT), two trans-Golgi glycosyltransferases, with respect to their different pathways in monensin-treated cells. Upon addition of monensin galT dissociates from siaT and the GA and accumulates in swollen vesicles derived from the trans-Golgi network (TGN), as shown by colocalization with TGN46, a specific TGN marker. We analyzed various chimeric constructs of galT and siaT by confocal fluorescence microscopy and time-lapse videomicroscopy as well as Optiprep density gradient fractionation. We show that the first 13 amino acids of the cytoplasmic tail of galT are necessary for its localization to swollen vesicles induced by monensin. We also show that the monensin sensitivity resulting from the cytoplasmic tail can be conferred to siaT, which leads to the rapid accumulation of the galT–siaT chimera in swollen vesicles upon monensin treatment. On the basis of these data, we suggest that cycling between the trans-Golgi cisterna and the trans-Golgi network of galT is signal mediated.

BFA-induced compartments from the Golgi apparatus and trans-Golgi network/early endosome are distinct in plant cells

2009 ◽  
Vol 60 (5) ◽  
pp. 865-881 ◽  
Author(s):  
Sheung Kwan Lam ◽  
Yi Cai ◽  
Yu Chung Tse ◽  
Juan Wang ◽  
Angus Ho Yin Law ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Plant Cells ◽  
Early Endosome ◽  
Trans Golgi Network ◽  
Golgi Network

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 Glucose regulates clathrin adaptors at the trans-Golgi network and endosomes

2011 ◽  
Vol 22 (19) ◽  
pp. 3671-3683 ◽  
Author(s):  
Quyen L. Aoh ◽  
Lee M. Graves ◽  
Mara C. Duncan
Keyword(s):  
Cell Polarity ◽  
Translation Initiation ◽  
Posttranslational Modifications ◽  
Raw Material ◽  
Eukaryotic Cells ◽  
Glucose Starvation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Trans Golgi Network ◽  
Golgi Network ◽  
Biomass Increase

Glucose is a rich source of energy and the raw material for biomass increase. Many eukaryotic cells remodel their physiology in the presence and absence of glucose. The yeast Saccharomyces cerevisiae undergoes changes in transcription, translation, metabolism, and cell polarity in response to glucose availability. Upon glucose starvation, translation initiation and cell polarity are immediately inhibited, and then gradually recover. In this paper, we provide evidence that, as in cell polarity and translation, traffic at the trans-Golgi network (TGN) and endosomes is regulated by glucose via an unknown mechanism that depends on protein kinase A (PKA). Upon glucose withdrawal, clathrin adaptors exhibit a biphasic change in localization: they initially delocalize from the membrane within minutes and later partially recover onto membranes. Additionally, the removal of glucose induces changes in posttranslational modifications of adaptors. Ras and Gpr1 signaling pathways, which converge on PKA, are required for changes in adaptor localization and changes in posttranslational modifications. Acute inhibition of PKA demonstrates that inhibition of PKA prior to glucose withdrawal prevents several adaptor responses to starvation. This study demonstrates that PKA activity prior to glucose starvation primes membrane traffic at the TGN and endosomes in response to glucose starvation.

Benzyl alcohol induces a reversible fragmentation of the Golgi apparatus and inhibits membrane trafficking between endosomes and the trans-Golgi network

2017 ◽  
Vol 357 (1) ◽  
pp. 67-78 ◽  
Author(s):  
Roger Simm ◽  
Audun Sverre Kvalvaag ◽  
Bo van Deurs ◽  
Toril Lindbäck ◽  
Kirsten Sandvig
Keyword(s):  
Golgi Apparatus ◽  
Benzyl Alcohol ◽  
Membrane Trafficking ◽  
Trans Golgi Network ◽  
Golgi Network

scholarly journals Energy metabolism regulates clathrin adaptors at the trans-Golgi network and endosomes

2013 ◽  
Vol 24 (6) ◽  
pp. 832-847 ◽  
Author(s):  
Quyen L. Aoh ◽  
Chao-wei Hung ◽  
Mara C. Duncan
Keyword(s):  
Energy Metabolism ◽  
Intracellular Signaling ◽  
Phosphatidyl Inositol ◽  
Glucose Starvation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Permeabilized Cells ◽  
Trans Golgi Network ◽  
Golgi Network ◽  
Energy Dependent ◽  
Adp Ribosylation Factor

Glucose is a master regulator of cell behavior in the yeast Saccharomyces cerevisiae. It acts as both a metabolic substrate and a potent regulator of intracellular signaling cascades. Glucose starvation induces the transient delocalization and then partial relocalization of clathrin adaptors at the trans-Golgi network and endosomes. Although these localization responses are known to depend on the protein kinase A (PKA) signaling pathway, the molecular mechanism of this regulation is unknown. Here we demonstrate that PKA and the AMP-regulated kinase regulate adaptor localization through changes in energy metabolism. We show that genetic and chemical manipulation of intracellular ATP levels cause corresponding changes in adaptor localization. In permeabilized cells, exogenous ATP is sufficient to induce adaptor localization. Furthermore, we reveal distinct energy-dependent steps in adaptor localization: a step that requires the ADP-ribosylation factor ARF, an ATP-dependent step that requires the phosphatidyl-inositol-4 kinase Pik1, and third ATP-dependent step for which we provide evidence but for which the mechanism is unknown. We propose that these energy-dependent mechanisms precisely synchronize membrane traffic with overall proliferation rates and contribute a crucial aspect of energy conservation during acute glucose starvation.

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