S. M. Karunaratne

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/

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 µm 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. Methodology/Principal Findings We exploited the tractability of macropinosomes through image-based screening and systematic overexpression of SNX-PX-BAR proteins to quantitate their effect on macropinosome formation. SNX1 (40.9+/−3.19 macropinosomes), SNX5 (36.99+/−4.48 macropinosomes), SNX9 (37.55+/−2.4 macropinosomes), SNX18 (88.2+/−8 macropinosomes), SNX33 (65.25+/−6.95 macropinosomes) all exhibited statistically significant (p<0.05) increases in average macropinosome numbers per 100 transfected cells as compared to control cells (24.44+/−1.81 macropinosomes). SNX1, SNX5, SNX9, and SNX18 were also found to associate with early-stage macropinosomes within 5 minutes following organelle formation. The modulation of intracellular PI(3,4,5)P3 levels through overexpression of PTEN or a lipid phosphatase-deficient mutant PTEN(G129E) was also observed to significantly reduce or elevate macropinosome formation respectively; coexpression of PTEN(G129E) with SNX9 or SNX18 synergistically elevated macropinosome formation to 119.4+/−7.13 and 91.4+/−6.37 macropinosomes respectively (p<0.05). Conclusions/Significance SNX1, SNX5, SNX9, SNX18, and SNX33 were all found to elevate macropinosome formation and (with the exception of SNX33) associate with early-stage macropinosomes. Moreover the effects of SNX9 and SNX18 overexpression in elevating macropinocytosis is likely to be synergistic with the increase in PI(3,4,5)P3 levels, which is known to accumulate on the cell surface and early-stage macropinocytic cups. Together these findings represent the first systematic functional study into the impact of the SNX-PX-BAR family on macropinocytosis.

Towards defining the nuclear proteome

BackgroundThe nucleus is a complex cellular organelle and accurately defining its protein content is essential before any systematic characterization can be considered.ResultsWe report direct evidence for 2,568 mammalian proteins within the nuclear proteome: the nuclear subcellular localization of 1,529 proteins based on a high-throughput subcellular localization protocol of full-length proteins and an additional 1,039 proteins for which clear experimental evidence is documented in published literature. This is direct evidence that the nuclear proteome consists of at least 14% of the entire proteome. This dataset was used to evaluate computational approaches designed to identify additional nuclear proteins.ConclusionThis represents direct experimental evidence that the nuclear proteome consists of at least 14% of the entire proteome. This high-quality nuclear proteome dataset was used to evaluate computational approaches designed to identify additional nuclear proteins. Based on this analysis, researchers can determine the stringency and types of lines of evidence they consider to infer the size and complement of the nuclear proteome.

Improved detection of lacZ reporter gene expression in transgenic epithelia by immunofluorescence microscopy

Abstract: The bacterial lacZ gene is commonly used as a reporter for the in vivo analysis of gene regulation in transgenic mice. However, several laboratories have reported poor detection of β‐galactosidase (the lacZ gene product) using histochemical techniques, particularly in skin. Here we report the difficulties we encountered in assessing lacZ expression in transgenic keratinocytes using classic X‐gal histochemical protocols in tissues shown to express the transgene by mRNA in situ hybridization. We found that lacZ reporter gene expression could be reliably detected in frozen tissue sections by immunofluorescence analysis using a β‐galactosidase‐specific antibody. Moreover, we were able to localize both transgene and endogenous gene products simultaneously using double‐label immunofluorescence. Our results suggest that antibody detection of β‐galactosidase should be used to verify other assays of lacZ expression, particularly where low expression levels are suspected or patchy expression is observed.