scholarly journals Analysis of membrane proteins localizing to the inner nuclear envelope in living cells

2016 ◽  
Vol 215 (4) ◽  
pp. 575-590 ◽  
Author(s):  
Christine J. Smoyer ◽  
Santharam S. Katta ◽  
Jennifer M. Gardner ◽  
Lynn Stoltz ◽  
Scott McCroskey ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Nuclear Membrane ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Transmembrane Protein ◽  
Protein Composition ◽  
Living Cells ◽  
Correlation Spectroscopy ◽  
Green Fluorescent ◽  
And Function

Understanding the protein composition of the inner nuclear membrane (INM) is fundamental to elucidating its role in normal nuclear function and in disease; however, few tools exist to examine the INM in living cells, and the INM-specific proteome remains poorly characterized. Here, we adapted split green fluorescent protein (split-GFP) to systematically localize known and predicted integral membrane proteins in Saccharomyces cerevisiae to the INM as opposed to the outer nuclear membrane. Our data suggest that components of the endoplasmic reticulum (ER) as well as other organelles are able to access the INM, particularly if they contain a small extraluminal domain. By pairing split-GFP with fluorescence correlation spectroscopy, we compared the composition of complexes at the INM and ER, finding that at least one is unique: Sbh2, but not Sbh1, has access to the INM. Collectively, our work provides a comprehensive analysis of transmembrane protein localization to the INM and paves the way for further research into INM composition and function.

PIT-1 Protein Localization at Different Optical Sections in a Single Living Cell Using FRET Microscopy and Green Fluorescent Proteins

1997 ◽  
Vol 3 (S2) ◽  
pp. 133-134 ◽  
Author(s):  
Ammasi Periasamy ◽  
Richard N. Day
Keyword(s):  
Transcription Factors ◽  
Living Cell ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Protein Localization ◽  
Resonance Energy ◽  
Living Cells ◽  
Specific Gene ◽  
Green Fluorescent ◽  
Prl Gene

The pituitary specific transcription factor Pit-1 is required for transcriptional activity of the prolactin (PRL) gene. The Pit-1 protein is a member of the POU homeodomain transcription factors that is expressed in several different anterior pituitary cell types, where it functions as an important determinant of pituitary-specific gene expression. The Pit-1 protein generally interacts with DNA elements in the PRL gene promoter as a dimer, and has been demonstrated to associate with other transcription factors. The objective of our research is to define the critical molecular events involved in transcriptional regulation of the PRL gene in living cells. Methods that allow monitoring of the intimate interactions between protein partners in living cells provide an unparalleled perspective on these biological processes. Using the jellyfish green fluorescent protein (GFP) as a tag, we applied the fluorescence resonance energy transfer (FRET) technique to visualize where and when the Pit-1 protein interacts in the living cell. FRET is a quantum mechanical effect that occurs between donor (D) and acceptor (A) fluorophores provided: (i) the emission energy of D is coincident with the energy required to excite A, and (ii) the distance that separating the two fluorophores is 10-100 Å. Mutant forms of GFP that fluoresce either green or blue (BFP) have excitation and emission spectra that are suitable for FRET imaging.

scholarly journals Ablation of a small transmembrane protein of Trypanosoma brucei (TbVTC1) involved in the synthesis of polyphosphate alters acidocalcisome biogenesis and function, and leads to a cytokinesis defect

2007 ◽  
Vol 407 (2) ◽  
pp. 161-170 ◽  
Author(s):  
Jianmin Fang ◽  
Peter Rohloff ◽  
Kildare Miranda ◽  
Roberto Docampo
Keyword(s):  
Trypanosoma Brucei ◽  
Fluorescent Protein ◽  
Polyclonal Antibodies ◽  
Transmembrane Protein ◽  
Immunogold Electron Microscopy ◽  
Protonmotive Force ◽  
Morphological Alterations ◽  
A Cell ◽  
Green Fluorescent ◽  
And Function

Inorganic poly P (polyphosphate) is an abundant component of acidocalcisomes of Trypanosoma brucei. In the present study we report the presence of a protein homologous with the yeast Vtc1p (vacuolar transporter chaperone 1) in T. brucei that is essential for poly P synthesis, acidocalcisome biogenesis and cytokinesis. Localization studies in a cell line expressing a TbVTC1 fused to GFP (green fluorescent protein) revealed its co-localization with the V-H+-PPase (vacuolar H+-pyrophosphatase), a marker for acidocalcisomes. Western blot analysis of acidocalcisome fractions and immunogold electron microscopy using polyclonal antibodies against a fragment of TbVTC1 confirmed the acidocalcisome localization. Ablation of TbVTC1 expression by RNA interference caused an abnormal morphology of acidocalcisomes, indicating that their biogenesis was disturbed, with a decreased pyrophosphate-driven H+ uptake and Ca2+ content, a significant decrease in the amount of poly P and a deficient response to hyposmotic stress. Ablation of TbVTC1 expression for longer periods produced marked gross morphological alterations compatible with a defect in cytokinesis, followed by cell death. Overexpression of the TbVTC1 gene caused mild alterations in growth rate, but had no perceptible effect on acidocalcisome morphology. We propose that the PPi-driven H+ pumping deficiency induced by ablation of TbVTC1 leads to alterations in the protonmotive force of acidocalcisomes, which results in deficient fusion or budding of the organelles, decreased H+ and Ca2+ content, and decreased synthesis of poly P. A decrease in the poly P content would lead to osmotic sensitivity and defects in cytokinesis.

scholarly journals New tools for the visualization of glial fibrillary acidic protein in living cells

2020 ◽  
Vol 1 ◽  
Author(s):  
Ricardo Letra-Vilela ◽  
Ricardo Quiteres ◽  
Fernanda Murtinheira ◽  
Alvaro Crevenna ◽  
Zach Hensel ◽  
...  
Keyword(s):  
Glial Fibrillary Acidic Protein ◽  
Fluorescent Protein ◽  
Living Cells ◽  
Alexander Disease ◽  
Acidic Protein ◽  
System A ◽  
The Central Nervous System ◽  
Green Fluorescent ◽  
And Function ◽  
Protein Tags

AbstractThe glial fibrillary acidic protein (GFAP) is an intermediate filament widely used to identify and label astroglial cells, a very abundant and relevant glial cell type in the central nervous system. A major hurdle in studying its behavior and function arises from the fact that GFAP does not tolerate well the addition of protein tags to its termini. Here, we tagged human GFAP (hGFAP) with an enhanced green fluorescent protein (EGFP) for the first time, and substituted a previously reported EGFP tag on mouse GFAP (mGFAP) by a more versatile Halo Tag. Both versions of tagged GFAP were able to incorporate into the normal GFAP filamentous network in glioma cells, and Alexander disease-related mutations or pharmacological disruption of microtubules and actin filaments interfered with GFAP dynamics. These new tools could provide new fruitful venues for the study of GFAP oligomerization, aggregation and dynamics in living cells.

scholarly journals A minimalist model to measure interactions between proteins and synaptic vesicles

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Eleonora Perego ◽  
Sofiia Reshetniak ◽  
Charlotta Lorenz ◽  
Christian Hoffmann ◽  
Dragomir Milovanović ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Synaptic Vesicles ◽  
Fluorescent Protein ◽  
Correlation Spectroscopy ◽  
Quantitative Studies ◽  
Synaptic Structure ◽  
Green Fluorescent ◽  
And Function ◽  
Presynaptic Protein

AbstractProtein dynamics in the synaptic bouton are still not well understood, despite many quantitative studies of synaptic structure and function. The complexity of the synaptic environment makes investigations of presynaptic protein mobility challenging. Here, we present an in vitro approach to create a minimalist model of the synaptic environment by patterning synaptic vesicles (SVs) on glass coverslips. We employed fluorescence correlation spectroscopy (FCS) to measure the mobility of monomeric enhanced green fluorescent protein (mEGFP)-tagged proteins in the presence of the vesicle patterns. We observed that the mobility of all eleven measured proteins is strongly reduced in the presence of the SVs, suggesting that they all bind to the SVs. The mobility observed in these conditions is within the range of corresponding measurements in synapses of living cells. Overall, our simple, but robust, approach should enable numerous future studies of organelle-protein interactions in general.

scholarly journals Co-Translational Insertion of Aquaporins into Liposome for Functional Analysis via an E. coli Based Cell-Free Protein Synthesis System

Cells ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 1325 ◽  
Author(s):  
Ke Yue ◽  
Tran Nam Trung ◽  
Yiyong Zhu ◽  
Ralf Kaldenhoff ◽  
Lei Kai
Keyword(s):  
Functional Analysis ◽  
Membrane Proteins ◽  
Fluorescent Protein ◽  
Water Channel ◽  
New Approach ◽  
E Coli ◽  
Straightforward Method ◽  
Lipid Environment ◽  
Green Fluorescent ◽  
Eukaryotic Organisms

Aquaporins are important and well-studied water channel membrane proteins. However, being membrane proteins, sample preparation for functional analysis is tedious and time-consuming. In this paper, we report a new approach for the co-translational insertion of two aquaporins from Escherichia coli and Nicotiana tabacum using the CFPS system. This was done in the presence of liposomes with a modified procedure to form homogenous proteo-liposomes suitable for functional analysis of water permeability using stopped-flow spectrophotometry. Two model aquaporins, AqpZ and NtPIP2;1, were successfully incorporated into the liposome in their active forms. Shifted green fluorescent protein was fused to the C-terminal part of AqpZ to monitor its insertion and status in the lipid environment. This new fast approach offers a fast and straightforward method for the functional analysis of aquaporins in both prokaryotic and eukaryotic organisms.

scholarly journals ADP-Ribosylation Factor (ARF) Interaction Is Not Sufficient for Yeast GGA Protein Function or Localization

2002 ◽  
Vol 13 (9) ◽  
pp. 3078-3095 ◽  
Author(s):  
Annette L. Boman ◽  
Paul D. Salo ◽  
Melissa J. Hauglund ◽  
Nicole L. Strand ◽  
Shelly J. Rensink ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Carboxypeptidase Y ◽  
Minor Role ◽  
Temperature Sensitive ◽  
Adp Ribosylation ◽  
And Function ◽  
Adp Ribosylation Factor

Golgi-localized γ-ear homology domain, ADP-ribosylation factor (ARF)-binding proteins (GGAs) facilitate distinct steps of post-Golgi traffic. Human and yeast GGA proteins are only ∼25% identical, but all GGA proteins have four similar domains based on function and sequence homology. GGA proteins are most conserved in the region that interacts with ARF proteins. To analyze the role of ARF in GGA protein localization and function, we performed mutational analyses of both human and yeast GGAs. To our surprise, yeast and human GGAs differ in their requirement for ARF interaction. We describe a point mutation in both yeast and mammalian GGA proteins that eliminates binding to ARFs. In mammalian cells, this mutation disrupts the localization of human GGA proteins. Yeast Gga function was studied using an assay for carboxypeptidase Y missorting and synthetic temperature-sensitive lethality between GGAs andVPS27. Based on these assays, we conclude that non-Arf-binding yeast Gga mutants can function normally in membrane trafficking. Using green fluorescent protein-tagged Gga1p, we show that Arf interaction is not required for Gga localization to the Golgi. Truncation analysis of Gga1p and Gga2p suggests that the N-terminal VHS domain and C-terminal hinge and ear domains play significant roles in yeast Gga protein localization and function. Together, our data suggest that yeast Gga proteins function to assemble a protein complex at the late Golgi to initiate proper sorting and transport of specific cargo. Whereas mammalian GGAs must interact with ARF to localize to and function at the Golgi, interaction between yeast Ggas and Arf plays a minor role in Gga localization and function.

scholarly journals Disparate Subcellular Localization Patterns of Pseudomonas aeruginosa Type IV Pilus ATPases Involved in Twitching Motility

2005 ◽  
Vol 187 (3) ◽  
pp. 829-839 ◽  
Author(s):  
Poney Chiang ◽  
Marc Habash ◽  
Lori L. Burrows
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Yellow Fluorescent Protein ◽  
Distribution Patterns ◽  
Opportunistic Pathogen ◽  
Twitching Motility ◽  
Polar Localization ◽  
Type Iv ◽  
And Function

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa expresses polar type IV pili (TFP), which are responsible for adhesion to various materials and twitching motility on surfaces. Twitching occurs by alternate extension and retraction of TFP, which arise from assembly and disassembly of pilin subunits at the base of the pilus. The ATPase PilB promotes pilin assembly, while the ATPase PilT or PilU or both promote pilin dissociation. Fluorescent fusions to two of the three ATPases (PilT and PilU) were functional, as shown by complementation of the corresponding mutants. PilB and PilT fusions localized to both poles, while PilU fusions localized only to the piliated pole. To identify the portion of the ATPases required for localization, sequential C-terminal deletions of PilT and PilU were generated. The conserved His and Walker B boxes were dispensable for polar localization but were required for twitching motility, showing that localization and function could be uncoupled. Truncated fusions that retained polar localization maintained their distinctive distribution patterns. To dissect the cellular factors involved in establishing polarity, fusion protein localization was monitored with a panel of TFP mutants. The localization of yellow fluorescent protein (YFP)-PilT and YFP-PilU was independent of the subunit PilA, other TFP ATPases, and TFP-associated proteins previously shown to be associated with the membrane or exhibiting polar localization. In contrast, YFP-PilB exhibited diffuse cytoplasmic localization in a pilC mutant, suggesting that PilC is required for polar localization of PilB. Finally, localization studies performed with fluorescent ATPase chimeras of PilT and PilU demonstrated that information responsible for the characteristic localization patterns of the ATPases likely resides in their N termini.

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