Vacuole membrane protein 1 marks endoplasmic reticulum subdomains enriched in phospholipid synthesizing enzymes and is required for phosphoinositide distribution

The multispanning membrane protein vacuole membrane protein 1 (VMP1) marks and regulates endoplasmic reticulum (ER)‐domains associated with diverse ER‐organelle membrane contact sites. A proportion of these domains associate with endosomes during their maturation and remodeling. We found that these VMP1 domains are enriched in choline/ethanolamine phosphotransferase and phosphatidylinositol synthase (PIS1), 2 ER enzymes required for the synthesis of various phospholipids. Interestingly, the lack of VMP1 impairs the formation of PIS1‐enriched ER domains, suggesting a role in the distribution of phosphoinositides. In fact, depletion of VMP1 alters the distribution of PtdIns4P and proteins involved in the trafficking of PtdIns4P. Consistently, in these conditions, defects were observed in endosome trafficking and maturation as well as in Golgi morphology. We propose that VMP1 regulates the formation of ER domains enriched in lipid synthesizing enzymes. These domains might be necessary for efficient distribution of PtdIns4P and perhaps other lipid species. These findings, along with previous reports that involved VMP1 in regulating PtdIns3P during autophagy, expand the role of VMP1 in lipid trafficking and explain the pleiotropic effects observed in VMP1‐deficient mammalian cells and other model systems.      

The sequences of MinE responsible for its subcellular localization analyzed by competitive binding method in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The subcellular localization of a protein is important for its proper function. Escherichia coli MinE is a small protein with clear subcellular localization, which provides a good model to study protein localization mechanism. In the present study, a series of recombinant minEs truncated in one end or in the middle regions, fused with egfp, was constructed, and these recombinant proteins could compete to function with the chromosomal MinE. Our results showed that the sequences related to the subcellular localization of MinE span several functional domains, demonstrating that MinE positioning in cells depends on multiple factors. The eGFP fusions with some truncated MinE from N-terminal resulted in different cell phenotypes and localization features, implying that these fusions can interfere chromosomal MinE’s function, similar to MinE^36–88 phenotype in the previous report. The amino acid in the region (32–48) is sensitive to change MinE conformation and influence its dimerization. Some truncated protein structure could be unstable. Thus, the MinE localization is prerequisite for its proper anti-MinCD function and some new features of MinE were demonstrated. This approach can be extended for subcellular localization research for other essential proteins.     

Cross‐talk between monocyte invasion and astrocyte proliferation regulates scarring in brain injury

Scar formation after brain injury is still poorly understood. To further elucidate such processes, here, we examine the interplay between astrocyte proliferation taking place predominantly at the vascular interface and monocyte invasion. Using genetic mouse models that decrease or increase reactive astrocyte proliferation, we demonstrate inverse effects on monocyte numbers in the injury site. Conversely, reducing monocyte invasion using CCR2^?/? mice causes a strong increase in astrocyte proliferation, demonstrating an intriguing negative cross‐regulation between these cell types at the vascular interface. CCR2^?/? mice show reduced scar formation with less extracellular matrix deposition, smaller lesion site and increased neuronal coverage. Surprisingly, the GFAP⁺ scar area in these mice is also significantly decreased despite increased astrocyte proliferation. Proteomic analysis at the peak of increased astrocyte proliferation reveals a decrease in extracellular matrix synthesizing enzymes in the injury sites of CCR2^?/? mice, highlighting how early key aspects of scar formation are initiated. Taken together, we provide novel insights into the cross‐regulation of juxtavascular proliferating astrocytes and invading monocytes as a crucial mechanism of scar formation upon brain injury.     

P2RY1/ALK3-Expressing Cells within the Adult Human Exocrine Pancreas Are BMP-7 Expandable and Exhibit Progenitor-like Characteristics

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Citrus tristeza virus co-opts glyceraldehyde 3-phosphate dehydrogenase for its infectious cycle by interacting with the viral-encoded protein p23

Key message

Citrus tristeza virus encodes a unique protein, p23, with multiple functional roles that include co-option of the cytoplasmic glyceraldehyde 3-phosphate dehydrogenase to facilitate the viral infectious cycle.

Abstract

The genome of citrus tristeza virus (CTV), genus Closterovirus family Closteroviridae, is a single-stranded (+) RNA potentially encoding at least 17 proteins. One (p23), an RNA-binding protein of 209 amino acids with a putative Zn-finger and some basic motifs, displays singular features: (i) it has no homologues in other closteroviruses, (ii) it accumulates mainly in the nucleolus and Cajal bodies, and in plasmodesmata, and (iii) it mediates asymmetric accumulation of CTV RNA strands, intracellular suppression of RNA silencing, induction of some CTV syndromes and enhancement of systemic infection when expressed as a transgene ectopically or in phloem-associated cells in several Citrus spp. Here, a yeast two-hybrid screening of an expression library of Nicotiana benthamiana (a symptomatic experimental host for CTV), identified a transducin/WD40 domain protein and the cytosolic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as potential host interactors with p23. Bimolecular fluorescence complementation corroborated the p23-GAPDH interaction in planta and showed that p23 interacts with itself in the nucleolus, Cajal bodies and plasmodesmata, and with GAPDH in the cytoplasm (forming aggregates) and in plasmodesmata. The latter interaction was preserved in a p23 deletion mutant affecting the C-terminal domain, but not in two others affecting the Zn-finger and one internal basic motif. Virus-induced gene silencing of GAPDH mRNA resulted in a decrease of CTV titer as revealed by real-time RT-quantitative PCR and RNA gel-blot hybridization. Thus, like other viruses, CTV seems to co-opt GAPDH, via interaction with p23, to facilitate its infectious cycle.

     

A novel Mr 77,000 protein of the XY body of mammalian spermatocytes: its localization in normal animals and in Searle’s translocation carriers

We describe a novel XY body protein of rat and mice pachytene spermatocytes called XY77. Biochemical characterization showed that protein XY77 (Mr 77,000; pH value 8.3) is present in meiotic but absent in postmeiotic stages of spermatogenesis. With the aid of an antibody against protein XY77 together with another specific for XY body-associated protein XY40 we also investigated the localization of these proteins in mice carrying Searle’s translocation, a reciprocal X-autosomal translocation. We show here that in these mice the distribution of both XY77 and XY40 is abnormal. Our results indicate that in Searle’s translocation alterations are not restricted to the translocated autosome, but also involve chromatin segments corresponding originally to the sex chromosomes X and Y. Received: 21 December 1996; in revised form: 1 February 1997 / Accepted: 15 February 1997     

XY body formation during rat spermatogenesis: an immunocytochemical study using antibodies against XY body-associated proteins

The process of formation of the XY body during meiotic prophase was investigated by immunocytochemistry on cryosections of pubertal rat testes using antibodies against three different XY body-associated proteins. Here we show that these proteins are detectable at only partially overlapping temporal windows. These findings provide the first evidence that the previously described morphological changes in the structure of the XY body that occur during meiotic prophase are accompanied by considerable changes in its protein composition. Received: 22 May 1997 / Accepted: 14 June 1997     

Nup84, A Novel Nucleoporin That Is Associated With CAN/Nup214 on the Cytoplasmic Face of the Nuclear Pore Complex

The short filaments extending from the cytoplasmic face of nuclear pore complexes are thought to contain docking sites for nuclear import substrates. One component of these filaments is the large O-linked glycoprotein CAN/Nup214. Immunoprecipitation studies carried out under nondenaturing conditions, and using a variety of antibodies, reveal a novel nonglycosylated nucleoporin, Nup84, that is tightly associated with CAN/Nup214. Consistent with such an association, Nup84 is found to be exposed on the cytoplasmic face of the nuclear pore complex. cDNA sequence analyses indicate that Nup84 contains neither the GLFG nor the XFXFG repeats that are a characteristic of a number of other nuclear pore complex proteins. Secondary structure predictions, however, suggest that Nup84 contains a coiled–coil COOH-terminal domain, a conclusion supported by the observation of significant sequence similarity between this region of the molecule and various members of the tropomyosin family. Mutagenesis and expression studies indicate that the putative coiled–coil domain is required for association with the cytoplasmic face of the nuclear pore complex, whereas it is the NH₂-terminal region of Nup84 that contains the site of interaction with CAN/Nup214. These findings suggest a model in which Nup84 may function in the attachment of CAN/Nup214 to the central framework of the nuclear pore complex. In this way, Nup84 could play a central role in the organization of the interface between the pore complex and the cytoplasm.