Investigating the Secretory Pathway of the Baculovirus-Insect Cell System Using a Secretory Green Fluorescent Protein

2000 ◽  
Vol 16 (5) ◽  
pp. 716-723 ◽  
Author(s):  
P.R. LeDuc ◽  
E.M. Whiteley ◽  
G. Bao ◽  
M.J. Betenbaugh
Keyword(s):  
Green Fluorescent Protein ◽  
Insect Cell ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Cell System ◽  
Green Fluorescent

Transport of virally expressed green fluorescent protein through the secretory pathway in tobacco leaves is inhibited by cold shock and brefeldin A

Planta ◽  
1999 ◽  
Vol 208 (3) ◽  
pp. 392-400 ◽  
Author(s):  
Petra Boevink ◽  
Barry Martin ◽  
Karl Oparka ◽  
Simon Santa Cruz ◽  
Chris Hawes
Keyword(s):  
Green Fluorescent Protein ◽  
Secretory Pathway ◽  
Cold Shock ◽  
Fluorescent Protein ◽  
Brefeldin A ◽  
Tobacco Leaves ◽  
Green Fluorescent

Transport Through the Secretory Pathway of VSVG Tagged With Green Fluorescent Protein: Role of Tubulovesicular Carriers and Microtubules

1997 ◽  
Vol 3 (S2) ◽  
pp. 139-140
Author(s):  
John Presley ◽  
Koret Hirschberg ◽  
Nelson Cole
Keyword(s):  
Green Fluorescent Protein ◽  
Membrane Transport ◽  
G Protein ◽  
Golgi Complex ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Dynamic Properties ◽  
Living Cells ◽  
Morphological Properties ◽  
Green Fluorescent

The ts045 mutant of VSV G protein has been used in numerous studies to identify biochemical and morphological properties of membrane transport, due to its reversible misfolding and retention in the ER at 40°C and ability to traffic out of the ER and into the Golgi complex upon temperature reduction to 32oC. The dynamic properties of membrane transport intermediates of the secretory pathway, including their lifetime and fate within cells, have not until now been explored due to the inability to follow transport in single living cells. Here, we attached green fluorescent protein to the cytoplasmic tail of VSV G protein in order to visualize ER-to-Golgi and Golgi-to-plasma membrane transport in living cells. VSVG-GFP expressed in Cos cells accumulated in the ER at 40°C and translocated to the Golgi complex when shifted to 32oC. Translocation of the protein was followed using time-lapse imaging of live cells on a confocal microscope. VSVG-GFP accumulated in tubulovesicular structures scattered throughout the cell upon shift from 40°C to 15°C for three hours.

Production of baculoviruses and expression of green fluorescent protein in immobilised Sf21 insect cell cultivation

2006 ◽  
Vol 29 (1-2) ◽  
pp. 55-61 ◽  
Author(s):  
Jeong Hwa Son ◽  
Rainer Buchholz ◽  
Jung-Keug Park ◽  
Sung Koo Kim
Keyword(s):  
Green Fluorescent Protein ◽  
Insect Cell ◽  
Fluorescent Protein ◽  
Cell Cultivation ◽  
Green Fluorescent

scholarly journals Oligomerization of green fluorescent protein in the secretory pathway of endocrine cells

2001 ◽  
Vol 360 (3) ◽  
pp. 645-649 ◽  
Author(s):  
Renu K. JAIN ◽  
Paul B. M. JOYCE ◽  
Miguel MOLINETE ◽  
Philippe A. HALBAN ◽  
Sven-Ulrik GORR
Keyword(s):  
Green Fluorescent Protein ◽  
Endocrine Cells ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Secretory Granules ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Reporter Protein ◽  
Regulated Secretory Pathway ◽  
Green Fluorescent

Green fluorescent protein (GFP) is used extensively as a reporter protein to monitor cellular processes, including intracellular protein trafficking and secretion. In general, this approach depends on GFP acting as a passive reporter protein. However, it was recently noted that GFP oligomerizes in the secretory pathway of endocrine cells. To characterize this oligomerization and its potential role in GFP transport, cytosolic and secretory forms of enhanced GFP (EGFP) were expressed in GH4C1 and AtT-20 endocrine cells. Biochemical analysis showed that cytosolic EGFP existed as a 27kDa monomer, whereas secretory forms of EGFP formed disulphide-linked oligomers. EGFP contains two cysteine residues (Cys49 and Cys71), which could play a role in this oligomerization. Site-directed mutagenesis of Cys49 and Cys71 showed that both cysteine residues were involved in disulphide interactions. Substitution of either cysteine residue resulted in a reduction or loss of oligomers, although dimers of the secretory form of EGFP remained. Mutation of these residues did not adversely affect the fluorescence of EGFP. EGFP oligomers were stored in secretory granules and secreted by the regulated secretory pathway in endocrine AtT-20 cells. Similarly, the dimeric mutant forms of EGFP were still secreted via the regulated secretory pathway, indicating that the higher-order oligomers were not necessary for sorting in AtT-20 cells. These results suggest that the oligomerization of EGFP must be considered when the protein is used as a reporter molecule in the secretory pathway.

scholarly journals Oligomerization of green fluorescent protein in the secretory pathway of endocrine cells

2001 ◽  
Vol 360 (3) ◽  
pp. 645 ◽  
Author(s):  
Renu K. JAIN ◽  
Paul B.M. JOYCE ◽  
Miguel MOLINETE ◽  
Philippe A. HALBAN ◽  
Sven-Ulrik GORR
Keyword(s):  
Green Fluorescent Protein ◽  
Endocrine Cells ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Green Fluorescent

scholarly journals The recombinant C-terminus of the human MUC2 mucin forms dimers in Chinese-hamster ovary cells and heterodimers with full-length MUC2 in LS 174T cells

2003 ◽  
Vol 372 (2) ◽  
pp. 335-345 ◽  
Author(s):  
Martin E. LIDELL ◽  
Malin E. V. JOHANSSON ◽  
Matthias MÖRGELIN ◽  
Noomi ASKER ◽  
James R. GUM ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Chinese Hamster Ovary ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Chinese Hamster ◽  
Full Length ◽  
Globular Domain ◽  
Wild Type ◽  
C Terminus ◽  
Green Fluorescent

The entire cDNA corresponding to the C-terminal cysteine-rich domain of the human MUC2 apomucin, after the serine- and threonine-rich tandem repeat, was expressed in Chinese-hamster ovary-K1 cells and in the human colon carcinoma cell line, LS 174T. The C-terminus was expressed as a fusion protein with the green fluorescent protein and mycTag sequences and the murine immunoglobulin κ-chain signal sequence to direct the protein to the secretory pathway. Pulse–chase studies showed a rapid conversion of the C-terminal monomer into a dimer in both Chinese-hamster ovary-K1 and LS 174T cells. Disulphide-bond-stabilized dimers secreted into the media of both cell lines had a higher apparent molecular mass compared with the intracellular forms. The MUC2 C-terminus was purified from the spent culture medium and visualized by molecular electron microscopy. The dimer nature of the molecule was visible clearly and revealed that each monomer was attached to the other by a large globular domain. Gold-labelled antibodies against the mycTag or green fluorescent protein revealed that these were localized to the ends opposite to the parts responsible for the dimerization. The C-terminus expressed in LS 174T cells formed heterodimers with the full-length wild-type MUC2, but not with the MUC5AC mucin, normally expressed in LS 174T cells. The homodimers of the MUC2 C-termini were secreted continuously from the LS 174T cells, but no wild-type MUC2 secretion has been observed from these cells. This suggests that the information for sorting the MUC2 mucin into the regulated secretory pathway in cells having this ability is present in parts other than the C-terminus of MUC2.

scholarly journals The Canonical Twin-Arginine Translocase Components Are Not Required for Secretion of Folded Green Fluorescent Protein from the Ancestral Strain of Bacillus subtilis

2014 ◽  
Vol 80 (10) ◽  
pp. 3219-3232 ◽  
Author(s):  
Anthony J. Snyder ◽  
Sampriti Mukherjee ◽  
J. Kyle Glass ◽  
Daniel B. Kearns ◽  
Suchetana Mukhopadhyay
Keyword(s):  
Bacillus Subtilis ◽  
Green Fluorescent Protein ◽  
Signal Peptide ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Secretion Systems ◽  
Content Type ◽  
Twin Arginine Translocase ◽  
Green Fluorescent ◽  
Ancestral Strain

ABSTRACTCellular processes, such as the digestion of macromolecules, phosphate acquisition, and cell motility, require bacterial secretion systems. InBacillus subtilis, the predominant protein export pathways are Sec (generalized secretory pathway) and Tat (twin-arginine translocase). Unlike Sec, which secretes unfolded proteins, the Tat machinery secretes fully folded proteins across the plasma membrane and into the medium. Proteins are directed for Tat-dependent export by N-terminal signal peptides that contain a conserved twin-arginine motif. Thus, utilizing the Tat secretion system by fusing a Tat signal peptide is an attractive strategy for the production and export of heterologous proteins. As a proof of concept, we expressed green fluorescent protein (GFP) fused to the PhoD Tat signal peptide in the laboratory and ancestral strains ofB. subtilis. Secretion of the Tat-GFP construct, as well as secretion of proteins in general, was substantially increased in the ancestral strain. Furthermore, our results show that secreted, fluorescent GFP could be purified directly from the extracellular medium. Nonetheless, export was not dependent on the known Tat secretion components or the signal peptide twin-arginine motif. We propose that the ancestral strain contains additional Tat components and/or secretion regulators that were abrogated following domestication.

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