Identification of two intracellular mechanisms leading to reduced expression of oncoretrovirus envelope glycoproteins at the cell surface

All retrovirus glycoproteins have a cytoplasmic domain that plays several roles in virus replication. We have determined whether and how the cytoplasmic domains of oncoretrovirus glycoproteins modulate their intracellular trafficking, by using chimeric proteins that combined the alpha-chain of the interleukin-2 receptor with the glycoprotein cytoplasmic domains of five oncoretroviruses: human T-cell leukemia virus type 1 (HTLV-1), Rous sarcoma virus (RSV), bovine leukemia virus (BLV), murine leukemia virus (MuLV), and Mason-Pfizer monkey virus (MPMV). All of these proteins were synthesized and matured in the same way as a control protein with no retrovirus cytoplasmic domain. However, the amounts of all chimeric proteins at the cell surface were smaller than that of the control protein. The protein appearing at and leaving the cell surface and endocytosis were measured in stable transfectants expressing the chimera. We identified two groups of proteins which followed distinct intracellular pathways. Group 1 included chimeric proteins that reached the cell surface normally but were rapidly endocytosed afterwards. This group included the chimeric proteins with HTLV-1, RSV, and BLV cytoplasmic domains. Group 2 included chimeric proteins that were not detected at the cell surface, despite normal intracellular concentrations, and were accumulated in the Golgi complex. This group included the chimeric proteins with MuLV and MPMV cytoplasmic domains. Finally, we verified that the MuLV envelope glycoproteins behaved in the same way as the corresponding chimeras. These results indicate that retroviruses have evolved two distinct mechanisms to ensure a similar biological feature: low concentrations of their glycoproteins at the cell surface.     

Targeting by AutophaGy proteins (TAG): Targeting of IFNG-inducible GTPases to membranes by the LC3 conjugation system of autophagy

LC3 has been used as a marker to locate autophagosomes. However, it is also well established that LC3 can localize on various membranous structures other than autophagosomes. We recently demonstrated that the LC3 conjugation system (ATG7, ATG3, and ATG12–ATG5-ATG16L1) is required to target LC3 and IFNG (interferon, gamma)-inducible GTPases to the parasitophorus vacuole membrane (PVM) of a protist parasite Toxoplasma gondii and consequently for IFNG to control T. gondii infection. Here we show that not only LC3, but also its homologs (GABARAP, GABARAPL1, and GABARAPL2) localize on the PVM of T. gondii in a conjugation-dependent manner. Knockout/knockdown of all LC3 homologs led to a significant reduction in targeting of the IFNG-inducible GTPases to the PVM of T. gondii and the IFNG-mediated control of T. gondii infection. Furthermore, when we relocated the ATG12–ATG5-ATG16L1 complex, which specifies the conjugation site of LC3 homologs, to alternative target membranes, the IFNG-inducible GTPases were targeted to the new target membranes rather than the PVM of T. gondii. These data suggest that the localization of LC3 homologs onto a membrane by the LC3 conjugation system is necessary and sufficient for targeting of the IFNG-inducible GTPases to the membrane, implying Targeting by AutophaGy proteins (TAG). Our data further suggest that the conjugation of ubiquitin-like LC3 homologs to the phospholipids of membranes may change the destiny of the membranes beyond degradation through lysosomal fusion, as the conjugation of ubiquitin to proteins changes the destiny of the proteins beyond proteasomal degradation.     

Internal micromorphology of the frons plate in females of the oriental hornet

Scanning electron microscopy and energy dispersive x-ray analysis (SEM/EDS) have been used to study the internal micromorphology of the frons plate in the Oriental hornet, Vespa orientalis. A conical shaped organ was described which is recessed into the frons plate and projects toward the interior of the acoustic box. The latter is located on the inner side of the frons plate. On the exterior of the conical region are observed aggregates containing Ca and Si, and a thin transparent membrane bearing a hole in its center. The innermost surface of the conical structure terminates bluntly as a convex lentiform tip, bearing a transparent oval-shaped window in its center. The conical organ, excepting the window, is enclosed in several layers of epithelium. The structure of this many-layered conical organ is highly complex; its numerous sub-structures and the possible role as a gravity sense organ are discussed. © 1993 Wiley-Liss, Inc.     

Poly(A) polymerase I of Escherichia coli: characterization of the catalytic domain, an RNA binding site and regions for the interaction with proteins involved in mRNA degradation

Poly(A) polymerase I (PAP I) of Escherichia coli is a member of the nucleotidyltransferase (Ntr) superfamily that includes the eukaryotic PAPs and all the known tRNA CCA-adding enzymes. Five highly conserved aspartic acids in the putative catalytic site of PAP I were changed to either alanine or proline, demonstrating their importance for polymerase activity. A glycine that is absolutely conserved in all Ntrs was also changed yielding a novel mutant protein in which ATP was wastefully hydrolysed in a primer-independent reaction. This is the first work to characterize the catalytic site of a eubacterial PAP and, despite the conservation of certain sequences, we predict that the overall architecture of the eukaryotic and eubacterial active sites is likely to be different. Binding sites for RNase E, a component of the RNA degradosome, and RNA were mapped by North-western and Far-western blotting using truncated forms of PAP I. Additional protein-protein interactions were detected between PAP I and CsdA, RhlE and SrmB, suggesting an unexpected connection between PAP I and these E. coli DEAD box RNA helicases. These results show that the functional organization of PAP I is similar to the eukaryotic PAPs with an N-terminal catalytic domain, a C-terminal RNA binding domain and sites for the interaction with other protein factors.     

Genetic diversity and mating system in a tropical tree,Carapa guianensis (Meliaceae)

Carapa guianensis Aublet. (Meliaceae) is an abundant, canopy tree species often exported as fine wood products. The populations in the Atlantic coastal plain of Costa Rica have undergone a wide variety of human-induced changes in distribution and abundance, including isolation due to deforestation. Using protein electrophoretic analysis of seeds, we investigated the genetic variability and population differentiation of nine populations and the outcrossing rate in two populations that differed in density by a factor of 50%. Average polymorphism (35%) and heterozygosity (0.12) across populations were relatively low in comparison with other tropical tree species. Only 4.6% of the total genetic variability could be attributed to population differentiation even though populations were separated by as much as 70 km. However, genetic differentiation did increase with distance between pairs of compared populations. There were no apparent differences in genetic variation between logged and unlogged forests. The outcrossing rate for each population was not significantly different from 1.0, but there was evidence of nonrandom mating in the lower density population. High levels of gene flow are probably maintained by seed dispersal as seeds travel well throughout flooded forests and rivers. High population density and synchronous flowering also contribute to high outcrossing rates. These genetic characteristics suggest that Carapa guianensis is a good candidate for natural forest management where it occurs at high density. However, current management practices need to account for the rapid rate of deforestation occurring around managed natural forest sites that will result in the elimination of intervening populations.