Estimating eukaryotic subcellular proteomes

ngLOC is an n-gram-based Bayesian classification method that can predict the localization of a protein sequence over ten distinct subcellular organelles.

BackgroundUnderstanding protein subcellular localization is a necessary component toward understanding the overall function of a protein. Numerous computational methods have been published over the past decade, with varying degrees of success. Despite the large number of published methods in this area, only a small fraction of them are available for researchers to use in their own studies. Of those that are available, many are limited by predicting only a small number of organelles in the cell. Additionally, the majority of methods predict only a single location for a sequence, even though it is known that a large fraction of the proteins in eukaryotic species shuttle between locations to carry out their function.FindingsWe present a software package and a web server for predicting the subcellular localization of protein sequences based on the ngLOC method. ngLOC is an n-gram-based Bayesian classifier that predicts subcellular localization of proteins both in prokaryotes and eukaryotes. The overall prediction accuracy varies from 89.8% to 91.4% across species. This program can predict 11 distinct locations each in plant and animal species. ngLOC also predicts 4 and 5 distinct locations on gram-positive and gram-negative bacterial datasets, respectively.ConclusionsngLOC is a generic method that can be trained by data from a variety of species or classes for predicting protein subcellular localization. The standalone software is freely available for academic use under GNU GPL, and the ngLOC web server is also accessible at http://ngloc.unmc.edu.

Gut granules are cell type‐specific lysosome‐related organelles found within the intestinal cells of Caenorhabditis elegans. To investigate the regulation of lysosome‐related organelle size, we screened for C. elegans mutants with substantially enlarged gut granules, identifying alleles of the vacuolar‐type H+‐ATPase and uridine‐5′‐monophosphate synthase (UMPS)‐1. UMPS‐1 catalyzes the conversion of orotic acid to UMP; this comprises the two terminal steps in de novo pyrimidine biosynthesis. Mutations in the orthologous human gene UMPS result in the rare genetic disease orotic aciduria. The umps‐1(−) mutation promoted the enlargement of gut granules to 250 times their normal size, whereas other endolysosomal organelles were not similarly affected. UMPS‐1::green fluorescent protein was expressed in embryonic and adult intestinal cells, where it was cytoplasmically localized and not obviously associated with gut granules. Whereas the umps‐1(−) mutant is viable, combination of umps‐1(−) with mutations disrupting gut granule biogenesis resulted in synthetic lethality. The effects of mutations in pyr‐1, which encodes the enzyme catalyzing the first three steps of de novo pyrimidine biosynthesis, did not phenotypically resemble those of umps‐1(−); instead, the synthetic lethality and enlargement of gut granules exhibited by the umps‐1(−) mutant was suppressed by pyr‐1(−). In a search for factors that mediate the enlargement of gut granules in the umps‐1(−) mutant, we identified WHT‐2, an ABCG transporter previously implicated in gut granule function. Our data suggest that umps‐1(−) leads to enlargement of gut granules through a build‐up of orotic acid. WHT‐2 possibly facilitates the increase in gut granule size of the umps‐1(−) mutant by transporting orotic acid into the gut granule and promoting osmotically induced swelling of the compartment.

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Caenorhabditis elegans gut granules are lysosome-related organelles with birefringent contents. mrp-4, which encodes an ATP-binding cassette (ABC) transporter homologous to mammalian multidrug resistance proteins, functions in the formation of gut granule birefringence. mrp-4(À) embryos show a delayed appearance of birefringent material in the gut granule but otherwise appear to form gut granules properly. mrp-4(1) activity is required for the extracellular mislocalization of birefringent material, bodylength retraction, and NaCl sensitivity, phenotypes associated with defective gut granule biogenesis exhibited by embryos lacking the activity of GLO-1/Rab38, a putative GLO-1 guanine nucleotide exchange factor GLO-4, and the AP-3 complex. Multidrug resistance protein (MRP)-4 localizes to the gut granule membrane, consistent with it playing a direct role in the transport of molecules that compose and/or facilitate the formation of birefringent crystals within the gut granule. However, MRP-4 is also present in oocytes and early embryos, and our genetic analyses indicate that its site of action in the formation of birefringent material may not be limited to just the gut granule in embryos. In a search for genes that function similarly to mrp-4(1), we identified WHT-2, another ABC transporter that acts in parallel to MRP-4 for the formation of birefringent material in the gut granule.