The glycoprotein cytoplasmic tail of Uukuniemi virus (Bunyaviridae) interacts with ribonucleoproteins and is critical for genome packaging

We have analyzed the importance of specific amino acids in the cytoplasmic tail of the glycoprotein G(N) for packaging of ribonucleoproteins (RNPs) into virus-like particles (VLPs) of Uukuniemi virus (UUK virus), a member of the Bunyaviridae family. In order to study packaging, we added the G(N)/G(C) glycoprotein precursor (p110) to a polymerase I-driven minigenome rescue system to generate VLPs that are released into the supernatant. These particles can infect new cells, and reporter gene expression can be detected. To determine the role of UUK virus glycoproteins in RNP packaging, we performed an alanine scan of the glycoprotein G(N) cytoplasmic tail (amino acids 1 to 81). First, we discovered three regions in the tail (amino acids 21 to 25, 46 to 50, and 71 to 81) which are important for minigenome transfer by VLPs. Further mutational analysis identified four amino acids that were important for RNP packaging. These amino acids are essential for the binding of nucleoproteins and RNPs to the glycoprotein without affecting the morphology of the particles. No segment-specific interactions between the RNA and the cytoplasmic tail could be observed. We propose that VLP systems are useful tools for analyzing protein-protein interactions important for packaging of viral genome segments, assembly, and budding of other members of the Bunyaviridae family.     

The human Nup107-160 nuclear pore subcomplex contributes to proper kinetochore functions

We previously demonstrated that a fraction of the human Nup107-160 nuclear pore subcomplex is recruited to kinetochores at the onset of mitosis. However, the molecular determinants for its kinetochore targeting and the functional significance of this localization were not investigated. Here, we show that the Nup107-160 complex interacts with CENP-F, but that CENP-F only moderately contributes to its targeting to kinetochores. In addition, we show that the recruitment of the Nup107-160 complex to kinetochores mainly depends on the Ndc80 complex. We further demonstrate that efficient depletion of the Nup107-160 complex from kinetochores, achieved either by combining siRNAs targeting several of its subunits excluding Seh1, or by depleting Seh1 alone, induces a mitotic delay. Further analysis of Seh1-depleted cells revealed impaired chromosome congression, reduced kinetochore tension and kinetochore-microtubule attachment defects. Finally, we show that the presence of the Nup107-160 complex at kinetochores is required for the recruitment of Crm1 and RanGAP1-RanBP2 to these structures. Together, our data thus provide the first molecular clues underlying the function of the human Nup107-160 complex at kinetochores.     

Protein phosphatase-1 is a novel regulator of the interaction between IRBIT and the inositol 1,4,5-trisphosphate receptor

IRBIT is an IP3R [IP3 (inositol 1,4,5-trisphosphate) receptor]-binding protein that competes with IP3 for binding to the IP3R. Phosphorylation of IRBIT is essential for the interaction with the IP3R. The unique N-terminal region of IRBIT, residues 1-104 for mouse IRBIT, contains a PEST (Pro-Glu-Ser-Thr) domain with many putative phosphorylation sites. In the present study, we have identified a well-conserved PP1 (protein phosphatase-1)-binding site preceeding this PEST domain which enabled the binding of PP1 to IRBIT both in vitro and in vivo. IRBIT emerged as a mediator of its own dephosphorylation by associated PP1 and, hence, as a novel substrate specifier for PP1. Moreover, IRBIT-associated PP1 specifically dephosphorylated Ser68 of IRBIT. Phosphorylation of Ser68 was required for subsequent phosphorylation of Ser71 and Ser74, but the latter two sites were not targeted by PP1. We found that phosphorylation of Ser71 and Ser74 were sufficient to enable inhibition of IP3 binding to the IP3R by IRBIT. Finally, we have shown that mutational inactivation of the docking site for PP1 on IRBIT increased the affinity of IRBIT for the IP3R. This pinpoints PP1 as a key player in the regulation of IP3R-controlled Ca2+ signals.     

A complex of catalytically inactive protein phosphatase-1 sandwiched between Sds22 and inhibitor-3

Protein Ser/Thr phosphatase-1 (PP1) associates with a host of proteins to form substrate-specific holoenzymes. Sds22 and Inhibitor-3 (I3) are two independently described ancient interactors of PP1. We show here by various approaches that Sds22 and I3 form a heterotrimeric complex with PP1, both in cell lysates and after purification. The stability of the complex depended on functional PP1 interaction sites in Sds22 and I3, indicating that PP1 is sandwiched between Sds22 and I3. Intriguingly, I3 could not be replaced in this complex by another PP1 interactor with the same PP1 binding motif. In vitro, Sds22 and I3 were potent inhibitors of PP1, but with only some substrates. The inhibition by Sds22 could be reproduced with synthetic Sds22 fragments comprising leucine-rich repeats (LRR) 2 and 5. Sds22 and LRR5 also slowly converted PP1 into a conformation that was inactive with all tested substrates. Cell lysates that were prepared under conditions that prevented the Sds22-induced inactivation of PP1 contained a catalytically inactive complex of Sds22, PP1, and I3, indicating that this complex exists in vivo. Therefore, our studies show that a pool of PP1 is complexly controlled by both Sds22 and I3.     

Arabidopsis GCP2 and GCP3 are part of a soluble gamma-tubulin complex and have nuclear envelope targeting domains

In higher plants, microtubules (MTs) are assembled in distinctive arrays in the absence of a defined organizing center. Three MT nucleation sites have been described: the nuclear surface, the cell cortex and cortical MT branch points. The Arabidopsis thaliana (At) genome contains putative orthologues encoding all the components of characterized mammalian nucleation complexes: gamma-tubulin and gamma-tubulin complex proteins GCP2 to GCP6. We have cloned the cDNA encoding AtGCP2, and show that gamma-tubulin, AtGCP2 and AtGCP3 are part of the same tandem affinity-purified complex and are present in a large membrane-associated complex. In addition, small soluble gamma-tubulin complexes of the size expected for a gamma-tubulin core complex are recruited to isolated nuclei. Using immunogold labelling, AtGCP3 is localized to both the nuclear envelope (NE) and the plasma membrane. To identify domains that could play a role in targeting complexes to these nucleation sites, truncated AtGCP2- and AtGCP3-green fluorescent protein fusion proteins were expressed in BY-2 cells. Several domains from AtGCP2 and AtGCP3 are capable of targeting fusions to the NE. We propose that regulated recruitment of soluble gamma-tubulin-containing complexes is responsible for nucleation at dispersed sites in plant cells and contributes to the formation and organization of the various MT arrays.     

Rab6 and the secretory pathway affect oocyte polarity in Drosophila

The Drosophila oocyte is a highly polarized cell. Secretion occurs towards restricted neighboring cells and asymmetric transport controls the localization of several mRNAs to distinct cortical compartments. Here, we describe a role for the Drosophila ortholog of the Rab6 GTPase, Drab6, in establishing cell polarity during oogenesis. We found that Drab6 localizes to Golgi and Golgi-derived membranes and interacts with BicD. We also provide evidence that Drab6 and BicD function together to ensure the correct delivery of secretory pathway components, such as the TGFalpha homolog Gurken, to the plasma membrane. Moreover, in the absence of Drab6, osk mRNA localization and the organization of microtubule plus-ends at the posterior of the oocyte were both severely affected. Our results point to a possible connection between Rab protein-mediated secretion, organization of the cytoskeleton and mRNA transport.     

Species abundance distributions: moving beyond single prediction theories to integration within an ecological framework

Species abundance distributions (SADs) follow one of ecology's oldest and most universal laws – every community shows a hollow curve or hyperbolic shape on a histogram with many rare species and just a few common species. Here, we review theoretical, empirical and statistical developments in the study of SADs. Several key points emerge. (i) Literally dozens of models have been proposed to explain the hollow curve. Unfortunately, very few models are ever rejected, primarily because few theories make any predictions beyond the hollow-curve SAD itself. (ii) Interesting work has been performed both empirically and theoretically, which goes beyond the hollow-curve prediction to provide a rich variety of information about how SADs behave. These include the study of SADs along environmental gradients and theories that integrate SADs with other biodiversity patterns. Central to this body of work is an effort to move beyond treating the SAD in isolation and to integrate the SAD into its ecological context to enable making many predictions. (iii) Moving forward will entail understanding how sampling and scale affect SADs and developing statistical tools for describing and comparing SADs. We are optimistic that SADs can provide significant insights into basic and applied ecological science.