Increased Redox-Sensitive Green Fluorescent Protein Reduction Potential in the Endoplasmic Reticulum following Glutathione-Mediated Dimerization

Biochemistry ◽  
2013 ◽  
Vol 52 (19) ◽  
pp. 3332-3345 ◽  
Author(s):  
Deboleena Dipak Sarkar ◽  
Sarah K. Edwards ◽  
Justin A. Mauser ◽  
Allen M. Suarez ◽  
Maxwell A. Serowoky ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Green Fluorescent Protein ◽  
Reduction Potential ◽  
Fluorescent Protein ◽  
Green Fluorescent ◽  
Protein Reduction

scholarly journals mCLCA4 ER processing and secretion requires luminal sorting motifs

2008 ◽  
Vol 295 (1) ◽  
pp. C279-C287 ◽  
Author(s):  
Chunlei Huan ◽  
Kai Su Greene ◽  
Bo Shui ◽  
Gwendolyn Spizz ◽  
Haitao Sun ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Chloride Transport ◽  
Proteolytic Processing ◽  
Regulatory Sequences ◽  
Cellular Processing ◽  
Green Fluorescent

Ca+-activated Cl− channel (CLCA) proteins are encoded by a family of highly related and clustered genes in mammals that are markedly upregulated in inflammation and have been shown to affect chloride transport. Here we describe the cellular processing and regulatory sequences underlying murine (m) CLCA4 proteins. The 125-kDa mCLCA4 gene product is cleaved to 90- and 40-kDa fragments, and the NH2- and COOH-terminal fragments are secreted, where they are found in cell media and associated with the plasma membrane. The 125-kDa full-length protein is only found in the endoplasmic reticulum (ER), and specific luminal diarginine retention and dileucine forward trafficking signals contained within the CLCA4 sequence regulate export from the ER and proteolytic processing. Mutation of the dileucine luminal sequences resulted in ER trapping of the immaturely glycosylated 125-kDa peptide, indicating that proteolytic cleavage occurs following recognition of the trafficking motifs. Moreover, the mutated dileucine and diarginine signal sequences directed processing of a secreted form of enhanced green fluorescent protein in a manner consistent with the effects on mCLCA4.

scholarly journals When a Day Makes a Difference. Interpreting Data from Endoplasmic Reticulum-Targeted Green Fluorescent Protein Fusions in Cells Grown in Suspension Culture

2002 ◽  
Vol 128 (2) ◽  
pp. 341-344 ◽  
Author(s):  
Staffan Persson ◽  
John Love ◽  
Pei-Lan Tsou ◽  
Dominique Robertson ◽  
William F. Thompson ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Green Fluorescent Protein ◽  
Suspension Culture ◽  
Fluorescent Protein ◽  
Green Fluorescent ◽  
In Cells

scholarly journals Green fluorescent protein-based monitoring of endoplasmic reticulum redox poise

2013 ◽  
Vol 4 ◽  
Author(s):  
Julia Birk ◽  
Thomas Ramming ◽  
Alex Odermatt ◽  
Christian Appenzeller-Herzog
Keyword(s):  
Endoplasmic Reticulum ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Redox Poise ◽  
Green Fluorescent

scholarly journals Rer1p, a Retrieval Receptor for Endoplasmic Reticulum Membrane Proteins, Is Dynamically Localized to the Golgi Apparatus by Coatomer

2001 ◽  
Vol 152 (5) ◽  
pp. 935-944 ◽  
Author(s):  
Ken Sato ◽  
Miyuki Sato ◽  
Akihiko Nakano
Keyword(s):  
Endoplasmic Reticulum ◽  
Green Fluorescent Protein ◽  
Membrane Proteins ◽  
Golgi Apparatus ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Endoplasmic Reticulum Membrane ◽  
Golgi Membrane ◽  
Green Fluorescent

Rer1p, a yeast Golgi membrane protein, is required for the retrieval of a set of endoplasmic reticulum (ER) membrane proteins. We present the first evidence that Rer1p directly interacts with the transmembrane domain (TMD) of Sec12p which contains a retrieval signal. A green fluorescent protein (GFP) fusion of Rer1p rapidly cycles between the Golgi and the ER. Either a lesion of coatomer or deletion of the COOH-terminal tail of Rer1p causes its mislocalization to the vacuole. The COOH-terminal Rer1p tail interacts in vitro with a coatomer complex containing α and γ subunits. These findings not only give the proof that Rer1p is a novel type of retrieval receptor recognizing the TMD in the Golgi but also indicate that coatomer actively regulates the function and localization of Rer1p.

scholarly journals Formation of stacked ER cisternae by low affinity protein interactions

2003 ◽  
Vol 163 (2) ◽  
pp. 257-269 ◽  
Author(s):  
Erik L. Snapp ◽  
Ramanujan S. Hegde ◽  
Maura Francolini ◽  
Francesca Lombardo ◽  
Sara Colombo ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Green Fluorescent Protein ◽  
Molecular Mechanism ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Living Cells ◽  
Cytoplasmic Domains ◽  
Membrane Structures ◽  
High Affinity ◽  
Green Fluorescent

The endoplasmic reticulum (ER) can transform from a network of branching tubules into stacked membrane arrays (termed organized smooth ER [OSER]) in response to elevated levels of specific resident proteins, such as cytochrome b(5). Here, we have tagged OSER-inducing proteins with green fluorescent protein (GFP) to study OSER biogenesis and dynamics in living cells. Overexpression of these proteins induced formation of karmellae, whorls, and crystalloid OSER structures. Photobleaching experiments revealed that OSER-inducing proteins were highly mobile within OSER structures and could exchange between OSER structures and surrounding reticular ER. This indicated that binding interactions between proteins on apposing stacked membranes of OSER structures were not of high affinity. Addition of GFP, which undergoes low affinity, antiparallel dimerization, to the cytoplasmic domains of non–OSER-inducing resident ER proteins was sufficient to induce OSER structures when overexpressed, but addition of a nondimerizing GFP variant was not. These results point to a molecular mechanism for OSER biogenesis that involves weak homotypic interactions between cytoplasmic domains of proteins. This mechanism may underlie the formation of other stacked membrane structures within cells.

Vcx1-D1 (M383I), the Vcx1 mutant with a calcineurin-independent vacuolar Ca2+/H+ exchanger activity, confers calcineurin-independent Mn2+ tolerance in Saccharomyces cerevisiae

2016 ◽  
Vol 62 (6) ◽  
pp. 475-484 ◽  
Author(s):  
Yunying Zhao ◽  
Huihui Xu ◽  
Yan Zhang ◽  
Linghuo Jiang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Green Fluorescent Protein ◽  
Signaling Pathway ◽  
Fluorescent Protein ◽  
Budding Yeast ◽  
Wild Type ◽  
Green Fluorescent ◽  
Calcineurin Signaling

The Vcx1-M1 mutant is known to confer calcineurin-dependent Mn2+ tolerance in budding yeast. Here, we demonstrate that another Vcx1 mutant, Vcx1-D1 with calcineurin-independent vacuolar Ca2+/H+ exchanger activity, confers calcineurin-independent Mn2+ tolerance. Unlike Vcx1-M1, the Mn2+ tolerance conferred by Vcx1-D1 is dependent on the presence of Pmr1 or Pmc1. The Pmr1-dependent Mn2+ tolerance of Vcx1-D1 requires the presence of calcineurin but not the functioning of the Ca2+/calcineurin signaling pathway. Similar to the wild-type Vcx1, C-terminally green fluorescent protein tagged Vcx1-D1 and Vcx1-M1 mutants localize to the endoplasmic reticulum instead of its normal vacuolar destination, but they remain functional in Ca2+ sensitivity and Mn2+ tolerance.

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