Engineering a novel multifunctional green fluorescent protein tag for a wide variety of protein research.

BACKGROUND: Genetically encoded tag is a powerful tool for protein research. Various kinds of tags have been developed: fluorescent proteins for live-cell imaging, affinity tags for protein isolation, and epitope tags for immunological detections. One of the major problems concerning the protein tagging is that many constructs with different tags have to be made for different applications, which is time- and resource-consuming. METHODOLOGY/PRINCIPAL FINDINGS: Here we report a novel multifunctional green fluorescent protein (mfGFP) tag which was engineered by inserting multiple peptide tags, i.e., octa-histidine (8xHis), streptavidin-binding peptide (SBP), and c-Myc tag, in tandem into a loop of GFP. When fused to various proteins, mfGFP monitored their localization in living cells. Streptavidin agarose column chromatography with the SBP tag successfully isolated the protein complexes in a native form with a high purity. Tandem affinity purification (TAP) with 8xHis and SBP tags in mfGFP further purified the protein complexes. mfGFP was clearly detected by c-Myc-specific antibody both in immunofluorescence and immuno-electron microscopy (EM). These findings indicate that mfGFP works well as a multifunctional tag in mammalian cells. The tag insertion was also successful in other fluorescent protein, mCherry. CONCLUSIONS AND SIGNIFICANCE: The multifunctional fluorescent protein tag is a useful tool for a wide variety of protein research, and may have the advantage over other multiple tag systems in its higher expandability and compatibility with existing and future tag technologies

Membrane microdomain switching: a regulatory mechanism of amyloid precursor protein processing

Neuronal activity has an impact on β cleavage of amyloid precursor protein (APP) by BACE1 to generate amyloid-β peptide (Aβ). However, the molecular mechanisms underlying this effect remain to be elucidated. Cholesterol dependency of β cleavage prompted us to analyze immunoisolated APP-containing detergent-resistant membranes from rodent brains. We found syntaxin 1 as a key molecule for activity-dependent regulation of APP processing in cholesterol-dependent microdomains. In living cells, APP associates with syntaxin 1–containing microdomains through X11–Munc18, which inhibits the APP–BACE1 interaction and β cleavage via microdomain segregation. Phosphorylation of Munc18 by cdk5 causes a shift of APP to BACE1-containing microdomains. Neuronal hyperactivity, implicated in Aβ overproduction, promotes the switching of APP microdomain association as well as β cleavage in a partially cdk5-dependent manner. We propose that microdomain switching is a mechanism of cholesterol- and activity-dependent regulation of APP processing in neurons

Engineering a novel multifunctional green fluorescent protein tag for a wide variety of protein research.

BACKGROUND: Genetically encoded tag is a powerful tool for protein research. Various kinds of tags have been developed: fluorescent proteins for live-cell imaging, affinity tags for protein isolation, and epitope tags for immunological detections. One of the major problems concerning the protein tagging is that many constructs with different tags have to be made for different applications, which is time- and resource-consuming. METHODOLOGY/PRINCIPAL FINDINGS: Here we report a novel multifunctional green fluorescent protein (mfGFP) tag which was engineered by inserting multiple peptide tags, i.e., octa-histidine (8xHis), streptavidin-binding peptide (SBP), and c-Myc tag, in tandem into a loop of GFP. When fused to various proteins, mfGFP monitored their localization in living cells. Streptavidin agarose column chromatography with the SBP tag successfully isolated the protein complexes in a native form with a high purity. Tandem affinity purification (TAP) with 8xHis and SBP tags in mfGFP further purified the protein complexes. mfGFP was clearly detected by c-Myc-specific antibody both in immunofluorescence and immuno-electron microscopy (EM). These findings indicate that mfGFP works well as a multifunctional tag in mammalian cells. The tag insertion was also successful in other fluorescent protein, mCherry. CONCLUSIONS AND SIGNIFICANCE: The multifunctional fluorescent protein tag is a useful tool for a wide variety of protein research, and may have the advantage over other multiple tag systems in its higher expandability and compatibility with existing and future tag technologies

TMEM30A is a candidate interacting partner for the β-carboxyl-terminal fragment of amyloid-β precursor protein in endosomes

<div><p>Although the aggregation of amyloid-β peptide (Aβ) clearly plays a central role in the pathogenesis of Alzheimer’s disease (AD), endosomal traffic dysfunction is considered to precede Aβ aggregation and trigger AD pathogenesis. A body of evidence suggests that the β-carboxyl-terminal fragment (βCTF) of amyloid-β precursor protein (APP), which is the direct precursor of Aβ, accumulates in endosomes and causes vesicular traffic impairment. However, the mechanism underlying this impairment remains unclear. Here we identified TMEM30A as a candidate partner for βCTF. TMEM30A is a subcomponent of lipid flippase that translocates phospholipids from the outer to the inner leaflet of the lipid bilayer. TMEM30A physically interacts with βCTF in endosomes and may impair vesicular traffic, leading to abnormally enlarged endosomes. APP traffic is also concomitantly impaired, resulting in the accumulation of APP-CTFs, including βCTF. In addition, we found that expressed BACE1 accumulated in enlarged endosomes and increased Aβ production. Our data suggested that TMEM30A is involved in βCTF-dependent endosome abnormalities that are related to Aβ overproduction.</p></div

Coexpression of TMEM30A and APP decreased sAPPβ production but increased FL-APP and APP-CTFs.

<p>A, B: COS-7 cells were transfected with APP and the wild-type or KKLN mutant of TMEM30A or BACE1. After 24-h transfection, the medium was replaced, and the cells were further incubated for 24 h. Immunoblot analyses of cell lysate (A) and medium (B) are shown. C–H: Quantitative analyses are shown as follows and compared with control: (C) FL-APP, (D) αCTF, (E) βCTF, (F) sAPPα, (G) sAPPβ, and (H) Aβ [independent experiments were performed four times (<i>n</i> = 4), mean ± SEM, *<i>P</i> < 0.05, **<i>P</i> < 0.01; N.S., no significant difference].</p

TMEM30A interacted with APP at the APP N-terminal domain and Aβ N-terminal sequence.

<p>A: Schematic depiction of deletion mutants of APP-Venus used in this study. TM; Transmembrane domain. B: COS-7 cells were transfected with mCherry-TMEM30A and the wild-type or deletion mutant of APP-Venus. After 24 h, the medium was replaced with a fresh medium containing a γ-secretase inhibitor (10 μM DAPT) and further incubated for 24 h. (B) Coimmunoprecipitation analysis of COS-7 cell lysates using control mouse IgG or GFP antibody (3E6) to precipitate APP-Venus. The arrowhead, arrow, and asterisks represent mature, immature mCherry-TMEM30A, and deletion mutants of APP-Venus, respectively.</p

Intracellular interaction between βCTF and TMEM30A.

<p>A: Schematic depiction of APP-CTFs and their specific antibodies. TM; Transmembrane domain. B: COS-7 cells were transfected with APP and CFP-TMEM30A, untagged TMEM30A, or BACE1. To segregate β1- and β11-CTFs, immunoblot analysis was performed using high-resolution electrophoresis. β1CTF was detected by 82E1, which is specific for β1 cleaved end. Because BACE1 activity competes with α-secretase [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200988#pone.0200988.ref032" target="_blank">32</a>], α′CTF appeared to be derived from α-secretase cleavage. C, D: COS-7 cells were transfected with APP and the wild-type or KKLN mutant of CFP-TMEM30A. (C) Coimmunoprecipitation analysis of COS-7 cell lysates using control mouse IgG or GFP antibody to precipitate CFP-TMEM30A. (D) Immunofluorescence analysis. Cells were labeled with APPC15 antibody (green). Scale bar: 20 μm. E: Coimmunoprecipitation analysis of COS-7 cells that were transfected with artificial βCTF, SC100, and wild-type or KKLN mutant of CFP-TMEM30A using control mouse IgG or GFP antibody.</p

Coexpression of TMEM30A and APP induces enlarged endosomes.

<p>A, B: COS-7 cells were transfected with APP-Venus and mCherry-TMEM30A. (A) Coexpression of APP and TMEM30A resulted in the redistribution of these proteins in enlarged vesicles. Scale bar: 20 μm. (B) The distribution of APP-containing vesicles in transfected cells classified by their size. The average number was evaluated by eight fields (×100, 8–11 cells), with counting in each experiment [independent experiment performed thrice (<i>n</i> = 3), mean ± SEM, *<i>P</i> < 0.05, ***<i>P</i> < 0.001]. Auto-thresholded images were processed using Image J. (C) Upper panel: COS-7 cells were transfected with mCherry-TMEM30A. Cells were labeled with anti-Rab5 antibody (green). Lower panel: COS-7 cells were transfected with APP-Venus and CFP-TMEM30A. Cells were labeled with early endosome marker, anti-Rab5 (red). Scale bar: 20 μm.</p