Subcellular proteomics for determining iron-limited remodeling of plastids in the model diatom Thalassiosira pseudonana (Bacillariophyta)

Diatoms are important primary producers in the world's oceans, yet their growth is constrained in large regions by low bioavailable iron (Fe). Low-Fe stress-induced limitation of primary production is due to requirements for Fe in components of essential metabolic pathways including photosynthesis and other chloroplast plastid functions. Studies have shown that under low-Fe stress, diatoms alter plastid-specific processes, including components of electron transport. These physiological changes suggest changes of protein content and in protein abundances within the diatom plastid. While in silico predictions provide putative information on plastid-localized proteins, knowledge of diatom plastid proteins remains limited in comparison to well-studied model photosynthetic organisms. To address this, we employed shotgun proteomics to investigate the proteome of subcellular plastid-enriched fractions from Thalassiosira pseudonana to gain a better understanding of how the plastid proteome is remodeled in response to Fe limitation. Using mass spectrometry-based peptide identification and quantification, we analyzed T. pseudonana grown under Fe-replete and -limiting conditions. Through these analyses, we inferred the relative quantities of each protein, revealing that Fe limitation regulates major metabolic pathways in the plastid, including the Calvin cycle. Additionally, we observed changes in the expression of light-harvesting proteins. In silico localization predictions of proteins identified in this plastid-enriched proteome allowed for an in-depth comparison of theoretical versus observed plastid-localization, providing evidence for the potential of additional protein import pathways into the diatom plastid.     

Pathways for compartmentalizing protein synthesis in eukaryotic cells: the template-partitioning model.

mRNAs encoding signal sequences are translated on endoplasmic reticulum (ER) -- bound ribosomes, whereas mRNAs encoding cytosolic proteins are translated on cytosolic ribosomes. The partitioning of mRNAs to the ER occurs by positive selection; cytosolic ribosomes engaged in the translation of signal-sequence-bearing proteins are engaged by the signal-recognition particle (SRP) pathway and subsequently trafficked to the ER. Studies have demonstrated that, in addition to the SRP pathway, mRNAs encoding cytosolic proteins can also be partitioned to the ER, suggesting that RNA partitioning in the eukaryotic cell is a complex process requiring the activity of multiple RNA-partitioning pathways. In this review, key findings on this topic are discussed, and the template-partitioning model, describing a hypothetical mechanism for RNA partitioning in the eukaryotic cell, is proposed.     

Poly(A)-Binding Protein 1 Partially Relocalizes to the Nucleus during Herpes Simplex Virus Type 1 Infection in an ICP27-Independent Manner and Does Not Inhibit Virus Replication

Infection of cells by herpes simplex virus type 1 (HSV-1) triggers host cell shutoff whereby mRNAs are degraded and cellular protein synthesis is diminished. However, virus protein translation continues because the translational apparatus in HSV-infected cells is maintained in an active state. Surprisingly, poly(A)-binding protein 1 (PABP1), a predominantly cytoplasmic protein that is required for efficient translation initiation, is partially relocated to the nucleus during HSV-1 infection. This relocalization occurred in a time-dependent manner with respect to virus infection. Since HSV-1 infection causes cell stress, we examined other cell stress inducers and found that oxidative stress similarly relocated PABP1. An examination of stress-induced kinases revealed similarities in HSV-1 infection and oxidative stress activation of JNK and p38 mitogen-activated protein (MAP) kinases. Importantly, PABP relocalization in infection was found to be independent of the viral protein ICP27. The depletion of PABP1 by small interfering RNA (siRNA) knockdown had no significant effect on viral replication or the expression of selected virus late proteins, suggesting that reduced levels of cytoplasmic PABP1 are tolerated during infection.The lytic replication cycle of herpes simplex virus type 1 (HSV-1) can be divided into three phases, immediate-early (IE), early (E), and late (L), that occur in a coordinated sequential gene expression program. IE proteins can regulate E and L gene expression, which produces proteins involved in DNA replication, capsid production, and virion assembly. HSV infection results in host cell shutoff to facilitate the efficient production of viral proteins. First, mRNA is degraded by the virion-associated vhs protein, and then ICP27, a multifunctional regulator of gene expression, inhibits pre-mRNA splicing. As most viral mRNAs are intronless, this abrogates the production of stable cellular mRNAs that can be exported to the cytoplasm and compete for translation with viral mRNAs (44).HSV mRNAs are capped and polyadenylated and so are translated via a normal cap-dependent mechanism. Translation initiation, during which translationally active ribosomes are assembled, is a tightly regulated process (21). Eukaryotic initiation factor 4F (eIF4F) (composed of eIF4E, eIF4G, and eIF4A) that binds the cap at the 5′ end of the mRNA promotes the recruitment of the 40S ribosomal subunit and associated factors, including eIF2-GTP initiator tRNA. The recognition of the start codon then promotes large ribosomal subunit joining. Poly(A)-binding protein 1 (PABP1), which binds and multimerizes on mRNA poly(A) tails, enhances translation initiation through interactions with the eIF4G component of the eIF4F cap-binding complex (20, 29, 32, 51) to circularize the mRNA in a “closed-loop” conformation (24). Key protein-RNA and protein-protein interactions in the translation initiation complex are strengthened by this PABP1-mediated circularization (12).HSV-1 maintains active viral translation in the face of host translational shutoff. Infection activates protein kinase R (PKR), which phosphorylates eIF2α, resulting in translation inhibition. However, HSV-1 ICP34.5 redirects protein phosphatase 1α to reverse eIF2α phosphorylation, abrogating the block to translation (17, 38). In addition, the HSV-1 US11 protein inhibits PKR and may also block PKR-mediated eIF2α phosphorylation (40, 42). HSV-1 infection also enhances eIF4F assembly in quiescent cells by the phosphorylation and proteasome-mediated degradation of the eIF4E-binding protein (4E-BP), which, when hypophosphorylated, can negatively regulate eIF4F complex formation (54). However, ICP6 may also contribute to eIF4F assembly by binding to eIF4G (55). Finally, ICP6 is required for Mnk-1 phosphorylation of eIF4E, but the mechanisms behind this remain unclear (54). ICP27 has also been implicated in translation regulation during HSV infection (6, 8, 10, 30) and may also activate p38 mitogen-activated protein (MAP) kinase that can phosphorylate eIF4E (16, 59).PABP1 appears to be a common cellular target of RNA and DNA viruses. PABP1 can undergo proteolysis, intracellular relocalization, or modification of its interaction with other translation factors in response to infection. For example, poliovirus induces host cell shutoff by cleaving PABP1, thus disrupting certain PABP1-containing complexes (28, 29). The rotavirus NSP3 protein can displace PABP1 from translation initiation complexes (41). However, NSP3 also interacts with a cellular protein, RoXaN, which is required to relocate PABP1 to the nucleus (13). Similarly, the Kaposi''s sarcoma herpesvirus (KSHV) SOX protein plays a role in relocating PABP1, its cofactor in cellular mRNA decay, to the nucleus (33). Although steady-state levels of PABP1 are highest in the cytoplasm of normal cells, where it has cytoplasmic functions, it is a nucleocytoplasmic shuttling protein (1). However, it is unclear how PABP1 enters or exits the nucleus, as it contains neither a canonical nuclear export nor an import signal.Here we describe the loss of PABP1 from cap-binding complexes and the partial relocation of PABP1 to the nucleus in HSV-1-infected cells in a time-dependent manner. Relocation is specific for PABP1, as other translation factors remained in the cytoplasm. Cells undergo stress during HSV-1 infection, and analysis of a variety of cell stresses revealed that PABP relocalization was also observed upon oxidative stress. Paxillin, a potential PABP1 nuclear chaperone, was phosphorylated, and the paxillin-PABP1 interaction was reduced during virus infection. However, the interaction was weak and cell type dependent, indicating that other effectors of PABP1 relocation in the infected cell must exist. Recently, the HSV-1 ICP27 protein was suggested to alter the PABP1 cellular location (6). However, infections with ICP27-null mutant viruses clearly demonstrated that ICP27 is not required for PABP1 nuclear relocation in the context of infection. Although HSV-1 mRNAs are translated by a normal cap-dependent mechanism known to be enhanced by PABP1, small interfering RNA (siRNA) knockdown of PABP1 indicated that at late times of infection, the translation of certain virus late proteins tolerates very low levels of PABP1.     

Heterotachy and tree building: a case study with plastids and eubacteria

The nature of heterotachy at the center of recent controversy over the relative performance of tree-building methods is different from the form of heterotachy that has been inferred in empirical studies. The latter have suggested that proportions of variable sites (p(var)) vary among orthologues and among paralogues. However, the strength of this inference, describing what may be one of the most important evolutionary properties of sequence data, has remained weak. Consequently, other models of sequence evolution have been proposed to explain some long-branch attraction (LBA) problems that could be attributed to differences in p(var). For an empirical case with plastid and eubacterial RNA polymerase sequences, we confirm using capture-recapture estimates and simulations that p(var) can differ among orthologues in anciently diverged evolutionary lineages. We find that parsimony and a least squares distance method that implements an overly simple model of sequence evolution are susceptible to LBA induced by this form of heterotachy. Although homogeneous maximum likelihood inference was found to be robust to model misspecification in our specific example, we caution against assuming that it will always be so.     

Peptidoglycan from Bacillus cereus Mediates Commensalism with Rhizosphere Bacteria from the Cytophaga-Flavobacterium Group

Previous research in our laboratory revealed that the introduction of Bacillus cereus UW85 can increase the populations of bacteria from the Cytophaga-Flavobacterium (CF) group of the Bacteroidetes phylum in the soybean rhizosphere, suggesting that these rhizosphere microorganisms have a beneficial relationship (G. S. Gilbert, J. L. Parke, M. K. Clayton, and J. Handelsman, Ecology 74:840-854, 1993). In the present study, we determined the frequency at which CF bacteria coisolated with B. cereus strains from the soybean rhizosphere and the mechanism by which B. cereus stimulates the growth of CF rhizosphere strains in root exudate media. In three consecutive years of sampling, CF strains predominated among coisolates obtained with B. cereus isolates from field-grown soybean roots. In root exudate media, the presence of B. cereus was required for CF coisolate strains to reach high population density. However, rhizosphere isolates from the phylum Proteobacteria grew equally well in the presence and absence of B. cereus, and the presence of CF coisolates did not affect the growth of B. cereus. Peptidoglycan isolated from B. cereus cultures stimulated growth of the CF rhizosphere bacterium Flavobacterium johnsoniae, although culture supernatant from B. cereus grown in root exudate media did not. These results suggest B. cereus and CF rhizosphere bacteria have a commensal relationship in which peptidoglycan produced by B. cereus stimulates the growth of CF bacteria.     

Comparison of a Salmonella typhimurium proteome defined by shotgun proteomics directly on an LTQ-FT and by proteome pre-fractionation on an LCQ-DUO.

Shotgun proteomics is rapidly becoming one of the most efficient and popular tools to examine protein expression in cells. Numerous laboratories now have a wide array of low- and high-performance mass spectrometry instrumentation necessary to complete proteome-wide projects. Often these laboratories have time and financial constraints that prohibit all projects from being conducted on high-performance state-of-the-art mass spectrometers. Here, we compare shotgun proteomic results using a direct 'lyse, digest and analyse' approach on a high-performance mass spectrometer (i.e. the LTQ-FT) with the results from a much lower-performance instrument (i.e. the LCQ-DUO) where, for the latter, various traditional protein pre-fractionation steps and gas-phase fractionation were used to increase the proteome coverage. Our results demonstrate that shotgun proteomic analyses conducted on the lower-performance LCQ-DUO mass spectrometer could adequately characterize a PhoP constitutive strain of Salmonella typhimurium if proteome pre-fractionation steps and gas-phase fractionation were included.     

Directed differentiation of dendritic cells from mouse embryonic stem cells

Dendritic cells (DCs) are uniquely capable of presenting antigen to naive T cells, either eliciting immunity [1] or ensuring self-tolerance [2]. This property identifies DCs as potential candidates for enhancing responses to foreign [3] and tumour antigens [4], and as targets for immune intervention in the treatment of autoimmunity and allograft rejection [1]. Realisation of their therapeutic potential would be greatly facilitated by a fuller understanding of the function of DC-specific genes, a goal that has frequently proven elusive because of the paucity of stable lines of DCs that retain their unique properties, and the inherent resistance of primary DCs to genetic modification. Protocols for the genetic manipulation of embryonic stem (ES) cells are, by contrast, well established [5], as is their capacity to differentiate into a wide variety of cell types in vitro, including many of hematopoietic origin [6]. Here, we report the establishment, from mouse ES cells, of long-term cultures of immature DCs that share many characteristics with macrophages, but acquire, upon maturation, the allostimulatory capacity and surface phenotype of classical DCs, including expression of CD11c, major histocompatibility complex (MHC) class II and co-stimulatory molecules. This novel source should prove valuable for the generation of primary, untransformed DCs in which candidate genes have been overexpressed or functionally ablated, while providing insights into the earliest stages of DC ontogeny.