An evolutionarily conserved bimodular domain anchors ZC3HC1 and its yeast homologue Pml39p to the nuclear basket.

The proteins ZC3HC1 and TPR are structural components of the nuclear basket (NB), a fibrillar structure attached to the nucleoplasmic side of the nuclear pore complex (NPC). ZC3HC1 initially binds to the NB in a TPR-dependent manner and can subsequently recruit additional TPR polypeptides to this structure. Here, we examined the molecular properties of ZC3HC1 that enable its initial binding to the NB and TPR. We report the identification and definition of a nuclear basket-interaction domain (NuBaID) of HsZC3HC1 that comprises two similarly built modules, both essential for binding the NB-resident TPR. We show that such a bimodular construction is evolutionarily conserved, which we further investigated in Dictyostelium discoideum and Saccharomyces cerevisiae. Presenting ScPml39p as the ZC3HC1 homologue in budding yeast, we show that the bimodular NuBaID of Pml39p is essential for binding to the yeast NB and its TPR homologues ScMlp1p and ScMlp2p, and we further demonstrate that Pml39p enables linkage between subpopulations of Mlp1p. We eventually delineate the common NuBaID of the human, amoebic, and yeast homologue as the defining structural entity of a unique protein not found in all but likely present in most taxa of the eukaryotic realm.

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ZC3HC1 Is a Novel Inherent Component of the Nuclear Basket, Resident in a State of Reciprocal Dependence with TPR.

The nuclear basket (NB) scaffold, a fibrillar structure anchored to the nuclear pore complex (NPC), is regarded as constructed of polypeptides of the coiled-coil dominated protein TPR to which other proteins can bind without contributing to the NB's structural integrity. Here we report vertebrate protein ZC3HC1 as a novel inherent constituent of the NB, common at the nuclear envelopes (NE) of proliferating and non-dividing, terminally differentiated cells of different morphogenetic origin. Formerly described as a protein of other functions, we instead present the NB component ZC3HC1 as a protein required for enabling distinct amounts of TPR to occur NB-appended, with such ZC3HC1-dependency applying to about half the total amount of TPR at the NEs of different somatic cell types. Furthermore, pointing to an NB structure more complex than previously anticipated, we discuss how ZC3HC1 and the ZC3HC1-dependent TPR polypeptides could enlarge the NB's functional repertoire.

Type I Phosphatidylinositol-4-Phosphate 5-Kinases α and γ Play a Key Role in Targeting HIV-1 Pr55Gag to the Plasma Membrane.

HIV-1 assembly occurs principally at the plasma membrane (PM) of infected cells. Gag polyprotein precursors (Pr55Gag) are targeted to the PM, and their binding is mediated by the interaction of myristoylated matrix domain and a PM-specific phosphoinositide, the phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2]. The major synthesis pathway of PI(4,5)P2 involves the activity of phosphatidylinositol-4-phosphate 5-kinase family type 1 composed of three isoforms (PIP5K1α, PIP5K1ß, and PIP5K1γ). To examine whether the activity of a specific PIP5K1 isoform determines proper Pr55Gag localization at the PM, we compared the cellular behavior of Pr55Gag in the context of PIP5K1 inhibition using siRNAs that individually targeted each of the three isoforms in TZM-bl HeLa cells. We found that downregulation of PIP5K1α and PIP5K1γ strongly impaired the targeting of Pr55Gag to the PM with a rerouting of the polyprotein within intracellular compartments. The efficiency of Pr55Gag release was thus impaired through the silencing of these two isoforms, while PIP5K1ß is dispensable for Pr55Gag targeting to the PM. The PM mistargeting due to the silencing of PIP5K1α leads to Pr55Gag hydrolysis through lysosome and proteasome pathways, while the silencing of PIP5K1γ leads to Pr55Gag accumulation in late endosomes. Our findings demonstrated that, within the PIP5K1 family, only the PI(4,5)P2 pools produced by PIP5K1α and PIP5K1γ are involved in the Pr55Gag PM targeting process.IMPORTANCE PM specificity of Pr55Gag membrane binding is mediated through the interaction of PI(4,5)P2 with the matrix (MA) basic residues. It was shown that overexpression of a PI(4,5)P2-depleting enzyme strongly impaired PM localization of Pr55Gag However, cellular factors that control PI(4,5)P2 production required for Pr55Gag-PM targeting have not yet been characterized. In this study, by individually inhibiting PIP5K1 isoforms, we elucidated a correlation between PI(4,5)P2 metabolism pathways mediated by PIP5K1 isoforms and the targeting of Pr55Gag to the PM of TZM-bl HeLa cells. Confocal microscopy analyses of cells depleted from PIP5K1α and PIP5K1γ show a rerouting of Pr55Gag to various intracellular compartments. Notably, Pr55Gag is degraded by the proteasome and/or by the lysosomes in PIP5K1α-depleted cells, while Pr55Gag is targeted to endosomal vesicles in PIP5K1γ-depleted cells. Thus, our results highlight, for the first time, the roles of PIP5K1α and PIP5K1γ as determinants of Pr55Gag targeting to the PM.

Protein Tpr is required for establishing nuclear pore-associated zones of heterochromatin exclusion.

Amassments of heterochromatin in somatic cells occur in close contact with the nuclear envelope (NE) but are gapped by channel- and cone-like zones that appear largely free of heterochromatin and associated with the nuclear pore complexes (NPCs). To identify proteins involved in forming such heterochromatin exclusion zones (HEZs), we used a cell culture model in which chromatin condensation induced by poliovirus (PV) infection revealed HEZs resembling those in normal tissue cells. HEZ occurrence depended on the NPC-associated protein Tpr and its large coiled coil-forming domain. RNAi-mediated loss of Tpr allowed condensing chromatin to occur all along the NE's nuclear surface, resulting in HEZs no longer being established and NPCs covered by heterochromatin. These results assign a central function to Tpr as a determinant of perinuclear organization, with a direct role in forming a morphologically distinct nuclear sub-compartment and delimiting heterochromatin distribution.

Nucleoporins as components of the nuclear pore complex core structure and Tpr as the architectural element of the nuclear basket.

The vertebrate nuclear pore complex (NPC) is a macromolecular assembly of protein subcomplexes forming a structure of eightfold radial symmetry. The NPC core consists of globular subunits sandwiched between two coaxial ring-like structures of which the ring facing the nuclear interior is capped by a fibrous structure called the nuclear basket. By postembedding immunoelectron microscopy, we have mapped the positions of several human NPC proteins relative to the NPC core and its associated basket, including Nup93, Nup96, Nup98, Nup107, Nup153, Nup205, and the coiled coil-dominated 267-kDa protein Tpr. To further assess their contributions to NPC and basket architecture, the genes encoding Nup93, Nup96, Nup107, and Nup205 were posttranscriptionally silenced by RNA interference (RNAi) in HeLa cells, complementing recent RNAi experiments on Nup153 and Tpr. We show that Nup96 and Nup107 are core elements of the NPC proper that are essential for NPC assembly and docking of Nup153 and Tpr to the NPC. Nup93 and Nup205 are other NPC core elements that are important for long-term maintenance of NPCs but initially dispensable for the anchoring of Nup153 and Tpr. Immunogold-labeling for Nup98 also results in preferential labeling of NPC core regions, whereas Nup153 is shown to bind via its amino-terminal domain to the nuclear coaxial ring linking the NPC core structures and Tpr. The position of Tpr in turn is shown to coincide with that of the nuclear basket, with different Tpr protein domains corresponding to distinct basket segments. We propose a model in which Tpr constitutes the central architectural element that forms the scaffold of the nuclear basket.