LOCATE: a mammalian protein subcellular localization database

LOCATE is a curated, web-accessible database that houses data describing the membrane organization and subcellular localization of mouse and human proteins. Over the past 2 years, the data in LOCATE have grown substantially. The database now contains high-quality localization data for 20% of the mouse proteome and general localization annotation for nearly 36% of the mouse proteome. The proteome annotated in LOCATE is from the RIKEN FANTOM Consortium Isoform Protein Sequence sets which contains 58 128 mouse and 64 637 human protein isoforms. Other additions include computational subcellular localization predictions, automated computational classification of experimental localization image data, prediction of protein sorting signals and third party submission of literature data. Collectively, this database provides localization proteome for individual subcellular compartments that will underpin future systematic investigations of these regions. It is available at http://locate.imb.uq.edu.au

Towards defining the nuclear proteome

Direct evidence is reported for 2,568 mammalian proteins within the nuclear proteome, consisting of at least 14% of the entire proteome

The SNX-PX-BAR Family in Macropinocytosis: The Regulation of Macropinosome Formation by SNX-PX-BAR Proteins

Background: Macropinocytosis is an actin-driven endocytic process, whereby membrane ruffles fold back onto the plasma membrane to form large (> 0.2 mu m in diameter) endocytic organelles called macropinosomes. Relative to other endocytic pathways, little is known about the molecular mechanisms involved in macropinocytosis. Recently, members of the Sorting Nexin (SNX) family have been localized to the cell surface and early macropinosomes, and implicated in macropinosome formation. SNX-PX-BAR proteins form a subset of the SNX family and their lipid-binding (PX) and membrane-curvature sensing (BAR) domain architecture further implicates their functional involvement in macropinosome formation

Macropinosome formation screening assay validation.

<p>A: 24 hours post transfection, HEK-Flp-In cell monolayers were pulsed for 5 minutes with 100 µg/mL dextran (10,000 MW) conjugated to tetramethylrhodamine (dextran-TR) at 37°C. The samples were then washed in 4°C PBS, fixed in 4% PFA and imaged and processed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013763#s4" target="_blank">Materials and Methods</a>. Briefly Z-stack images comprising of 3×5 µm Z slices were merged into a single RGB image of transfected cells (green) stained with dextran-TR (red) (Ai). The red channel from the RGB image was isolated and converted to an 8-bit grayscale image (Aii). Dextran-positive macropinosomes were selected based on size (>0.5 µm in diameter) and fluorescent intensity (>100). Selected macropinosomes are shown in the foreground as black (Aiii). This binary image was then converted to a mask and superimposed onto the Green channel of the original RGB image to measure the green fluorescent intensity of the area occupied by each macropinosome in the image (Aiv). The particles with green fluorescence intensity higher than background signal (>20) were considered to be macropinosomes within a transfected cell, represented in green. Red particles represent discarded macropinosomes determined to be outside of a transfected cell. Scale  = 10 µm. B, C, D: HEK-Flp-In cell monolayers were either serum-starved for 16 hours and treated with dextran-TR in the presence or absence of 100 ng/mL EGF for 5 minutes at 37°C (B), treated with 1 mM amiloride or carrier (0.6% Methanol) for 30 minutes at 37°C before pulsing with dextran-TR (C), or transiently transfected with pEGFP-C1 or pEGFP-SNX5 before pulsing with dextran-TR (D). The samples in each case were assayed for macropinosome formation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013763#s4" target="_blank">Materials and Methods</a>, quantitating the mean number of macropinosomes/100 transfected cells over 3 replicates of 500 transfected cells for each condition. * denotes statistical significance (p<0.05) using the Student's T-test. Error bars denote Standard Error of the Mean (S.E.M).</p

The SNX-PX-BAR family is involved in macropinosome formation.

<p>HEK-Flp-In cells transiently overexpressing GFP-tagged members of the SNX-PX-BAR family were assayed for macropinosome formation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013763#s4" target="_blank">Materials and Methods</a>. A: Dextran-TR labeling of cells transfected with the specified constructs. Scale bar  = 10 µm. B: The mean number of macropinosomes/100 transfected cells was quantitated over 8 replicates of 500 transfected cells for each condition. * denotes statistical significance (p<0.05) using the Student's T-test, performing pairwise analyses relative to cells transfected with pEGFP-C1 alone. Error bars denote S.E.M.</p

SNX1, SNX5, SNX9 and SNX18 associate with early macropinosomes.

<p>A: HEK-Flp-In cells were pulsed with 100 µg/mL dextran (10,000 MW) conjugated to Alexa-647 (dextran-647) for 5 minutes before being transferred to 4°C and washed with 0.45 mM CaCl₂ 1 mM MgCl₂ PBS. The cells were then treated with 80 U/mL Streptolysin O for 5 minutes at 4°C to only permeabilize the plasma membrane before washing in 0.45 mM CaCl₂ 1 mM MgCl₂ PBS and incubating with 37°C PBS for 5 minutes. Following fixation with 4% PFA for 30 minutes at 4°C, cell monolayers are incubated with monoclonal and polyclonal antibodies against SNX1 and SNX5 respectively, followed by Alexa-488-conjugated goat-anti-mouse and Cy3-conjugated goat-anti-rabbit IgG secondary antibodies. B, C, and D: 24 hours post transfection, HEK-Flp-In cells transfected with pEGFP-SNX9 (B), pEGFP-SNX18 (C) and pEGFP-SNX33 (D) were pulsed with dextran-TR for 5 minutes at 37°C prior to fixation at 4°C in 4% PFA. Images were collected on an LSM 510 Meta confocal microscope. Scale bar  = 5 µm.</p

The SH3 domain of SNX18 is required for elevation of macropinosome formation.

<p>HEK-Flp-In cells transiently overexpressing pEGFP, pEGFP-SNX18, or pEGFP-ΔSH3-SNX18 were assayed for macropinosome formation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013763#s4" target="_blank">Materials and Methods</a>. The mean number of macropinosomes/100 transfected cells was quantitated over 3 replicates of 500 transfected cells for each condition. Error bars denote S.E.M.</p