Final Subcellular Localization Research Articles

Using Diatom and Apicomplexan Models to Study the Heme Pathway of Chromera velia.

Heme biosynthesis is essential for almost all living organisms. Despite its conserved function, the pathway’s enzymes can be located in a remarkable diversity of cellular compartments in different organisms. This location does not always reflect their evolutionary origins, as might be expected from the history of their acquisition through endosymbiosis. Instead, the final subcellular localization of the enzyme reflects multiple factors, including evolutionary origin, demand for the product, availability of the substrate, and mechanism of pathway regulation. The biosynthesis of heme in the apicomonad Chromera velia follows a chimeric pathway combining heme elements from the ancient algal symbiont and the host. Computational analyses using different algorithms predict complex targeting patterns, placing enzymes in the mitochondrion, plastid, endoplasmic reticulum, or the cytoplasm. We employed heterologous reporter gene expression in the apicomplexan parasite Toxoplasma gondii and the diatom Phaeodactylum tricornutum to experimentally test these predictions. 5-aminolevulinate synthase was located in the mitochondria in both transfection systems. In T. gondii, the two 5-aminolevulinate dehydratases were located in the cytosol, uroporphyrinogen synthase in the mitochondrion, and the two ferrochelatases in the plastid. In P. tricornutum, all remaining enzymes, from ALA-dehydratase to ferrochelatase, were placed either in the endoplasmic reticulum or in the periplastidial space.

Tools for the Recognition of Sorting Signals and the Prediction of Subcellular Localization of Proteins From Their Amino Acid Sequences.

At the time of translation, nascent proteins are thought to be sorted into their final subcellular localization sites, based on the part of their amino acid sequences (i.e., sorting or targeting signals). Thus, it is interesting to computationally recognize these signals from the amino acid sequences of any given proteins and to predict their final subcellular localization with such information, supplemented with additional information (e.g., k-mer frequency). This field has a long history and many prediction tools have been released. Even in this era of proteomic atlas at the single-cell level, researchers continue to develop new algorithms, aiming at accessing the impact of disease-causing mutations/cell type-specific alternative splicing, for example. In this article, we overview the entire field and discuss its future direction.

In silico analysis of different signal peptides to discover a panel of appropriate signal peptides for secretory production of Interferon-beta 1b in Escherichia coli.

Signal peptides (SPs) are one of the most important factors for suitable secretion of the recombinant heterologous proteins in Escherichia coli (E. coli). The objective of this study was to identify a panel of signal peptides (among the 90 biologically active SPs) required for the secretory production of interferon-beta 1b (IFN-beta 1b) recombinant protein into the periplasmic space of E. coli host. In the initial step, after predicting the accurate locations of the cleavage sites of signal peptides and their discrimination scores using SignalP 4.1 server, 31 SPs were eliminated from further analysis because their discrimination scores were less than 0.5 or their cleavage sites were inappropriately located. Therefore, only 59 SPs could be theoretically applied to secrete IFN-beta 1b into the periplasmic space of E. coli. The physico-chemical and the solubility properties, which are necessary parameters for selecting appropriate SPs, were predicted using ProtParam and SOLpro servers using the 59 remaining signal peptides. The final subcellular localization of IFN-beta 1b in combination with different SPs was predicted using ProtComB server. Consequently, according to the ranking of 59 confirmed SPs, the obtained results revealed that SPs Flagellar P-ring protein (flgI), Glucan 1,3-beta-glucosidase I/II (EXG1) and outer membrane protein C (OmpC) were theoretically the most potent and desirable SPs for secretion of recombinant IFN-beta 1b into the periplasmic space of E. coli. For further studies in the future, the experimental investigations on the obtained results will be considered.

Protein trafficking defects in inherited kidney diseases

The nephron, the basic structural and functional unit of the kidney, is lined by different, highly differentiated polarized epithelial cells. Their concerted action modifies the composition of the glomerular ultrafiltrate through reabsorption and secretion of essential solutes to finally produce urine. The highly specialized properties of the different epithelial cell types of the nephron are remarkable and rely on the regulated delivery of specific proteins to their final subcellular localization. Hence, mutations affecting sorting of individual proteins or inactivating the epithelial trafficking machinery have severe functional consequences causing disease. The presence of mutations leading to protein trafficking defect is indeed a mechanism of pathogenesis seen in an increasing number of disorders, including about one-third of monogenic diseases affecting the kidney. In this review, we focus on representative diseases to discuss different molecular mechanisms that primarily lead to defective protein transport, such as endoplasmic reticulum retention, mistargeting, defective endocytosis or degradation, eventually resulting in epithelial cell and kidney dysfunction. For each disease, we discuss the type of reported mutations, their molecular and cellular consequences and possible strategies for therapeutic intervention. Particular emphasis is given to new and prospective therapies aimed at rescuing the trafficking defect at the basis of these conformational diseases.

ATP9B, a P4-ATPase (a Putative Aminophospholipid Translocase), Localizes to the trans-Golgi Network in a CDC50 Protein-independent Manner

Type IV P-type ATPases (P4-ATPases) are putative phospholipid flippases that translocate phospholipids from the exoplasmic (lumenal) to the cytoplasmic leaflet of lipid bilayers and are believed to function in complex with CDC50 proteins. In Saccharomyces cerevisiae, five P4-ATPases are localized to specific cellular compartments and are required for vesicle-mediated protein transport from these compartments, suggesting a role for phospholipid translocation in vesicular transport. The human genome encodes 14 P4-ATPases and three CDC50 proteins. However, the subcellular localization of human P4-ATPases and their interactions with CDC50 proteins are poorly understood. Here, we show that class 5 (ATP10A, ATP10B, and ATP10D) and class 6 (ATP11A, ATP11B, and ATP11C) P4-ATPases require CDC50 proteins, primarily CDC50A, for their exit from the endoplasmic reticulum (ER) and final subcellular localization. In contrast, class 2 P4-ATPases (ATP9A and ATP9B) are able to exit the ER in the absence of exogenous CDC50 expression: ATP9B, but not ATP11B, was able to exit the ER despite depletion of CDC50 proteins by RNAi. Although ATP9A and ATP9B show a high overall sequence similarity, ATP9A localizes to endosomes and the trans-Golgi network (TGN), whereas ATP9B localizes exclusively to the TGN. A chimeric ATP9 protein in which the N-terminal cytoplasmic region of ATP9A was replaced with the corresponding region of ATP9B was localized exclusively to the Golgi. These results indicate that ATP9B is able to exit the ER and localize to the TGN independently of CDC50 proteins and that this protein contains a Golgi localization signal in its N-terminal cytoplasmic region.

Time and space resolved uptake study of silicananoparticles by human cells

A spatio-temporal mapping of the uptake of silica (SiO(2)) nanoparticles of different sizes by lung epithelial cells has been obtained. Based on high control of nanoparticle dispersion in cell media and cell exposure, one obtains reproducible and quantitative time-resolved data using a combination of flow cytometry, fluorescence and electron microscopies. We are thereby able to give a rather detailed account of the journey of SiO(2) nanoparticles from the early events of uptake to their final sub-cellular localization.

The N Terminus of Phosphodiesterase TbrPDEB1 of Trypanosoma brucei Contains the Signal for Integration into the Flagellar Skeleton

The precise subcellular localization of the components of the cyclic AMP (cAMP) signaling pathways is a crucial aspect of eukaryotic intracellular signaling. In the human pathogen Trypanosoma brucei, the strict control of cAMP levels by cAMP-specific phosphodiesterases is essential for parasite survival, both in cell culture and in the infected host. Among the five cyclic nucleotide phosphodiesterases identified in this organism, two closely related isoenzymes, T. brucei PDEB1 (TbrPDEB1) (PDEB1) and TbrPDEB2 (PDEB2) are predominantly responsible for the maintenance of cAMP levels. Despite their close sequence similarity, they are distinctly localized in the cell. PDEB1 is mostly located in the flagellum, where it forms an integral part of the flagellar skeleton. PDEB2 is mainly located in the cell body, and only a minor part of the protein localizes to the flagellum. The current study, using transfection of procyclic trypanosomes with green fluorescent protein (GFP) reporters, demonstrates that the N termini of the two enzymes are essential for determining their final subcellular localization. The first 70 amino acids of PDEB1 are sufficient to specifically direct a GFP reporter to the flagellum and to lead to its detergent-resistant integration into the flagellar skeleton. In contrast, the analogous region of PDEB2 causes the GFP reporter to reside predominantly in the cell body. Mutagenesis of selected residues in the N-terminal region of PDEB2 demonstrated that single amino acid changes are sufficient to redirect the reporter from a cell body location to stable integration into the flagellar skeleton.

Insight into the core and variant exoproteomes of Listeria monocytogenes species by comparative subproteomic analysis

While Listeria monocytogenes is responsible for listeriosis, it is also a saprophytic species with exceptional survival aptitudes. Secreted proteins are one of the main tools used by bacteria to interact with their environment. In order to take into account the biodiversity of L. monocytogenes species, exoproteomic analysis was carried out on 12 representative strains. Following 2-DE and MALDI-TOF MS, a total of 151 spots were identified and corresponded to 60 non-orthologous proteins. To categorize and analyze these proteomic data, a rational bioinformatic approach predicting final subcellular localization was carried out. Fifty-two out of the 60 proteins identified (86.7%) were indeed predicted as localized in the extracellular milieu (gene ontology (GO): 0005576). Most of them (65.4%) were actually predicted as secreted via the Sec translocon. Comparative analysis allowed for proteins found in all or only in a subset of L. monocytogenes strains to be defined. While the core exoproteome included most proteins related to bacterial virulence, cell wall biogenesis, as well as proteins secreted by unknown pathways, a slight variation in the protein members of these categories were observed and constituted the variant exoproteome. This investigation resulted in the first definition of the core and variant exoproteomes of L. monocytogenes where corollaries on bacterial physiology are further discussed.

Interaction of the Metalloprotease Disintegrins MDC9 and MDC15 with Two SH3 Domain-containing Proteins, Endophilin I and SH3PX1

Metalloprotease disintegrins (a disintegrin and metalloprotease (ADAM) and metalloprotease, disintegrin, cysteine-rich proteins (MDC)) are a family of membrane-anchored glycoproteins that function in diverse biological processes, including fertilization, neurogenesis, myogenesis, and ectodomain processing of cytokines and other proteins. The cytoplasmic domains of ADAMs often include putative signaling motifs, such as proline-rich SH3 ligand domains, suggesting that interactions with cytoplasmic proteins may affect metalloprotease disintegrin function. Here we report that two SH3 domain-containing proteins, endophilin I (SH3GL2, SH3p4) and a novel SH3 domain- and phox homology (PX) domain-containing protein, termed SH3PX1, can interact with the cytoplasmic domains of the metalloprotease disintegrins MDC9 and MDC15. These interactions were initially identified in a yeast two-hybrid screen and then confirmed using bacterial fusion proteins and co-immunoprecipitations from eukaryotic cells expressing both binding partners. SH3PX1 and endophilin I both preferentially bind the precursor but not the processed form of MDC9 and MDC15 in COS-7 cells. Since rat endophilin I is thought to play a role in synaptic vesicle endocytosis and SH3PX1 has sequence similarity to sorting nexins in yeast, we propose that endophilin I and SH3PX1 may have a role in regulating the function of MDC9 and MDC15 by influencing their intracellular processing, transport, or final subcellular localization.