Energy- and temperature-dependent transport of integral proteins to the inner nuclear membrane via the nuclear pore

Resident integral proteins of the inner nuclear membrane (INM) are synthesized as membrane-integrated proteins on the peripheral endoplasmic reticulum (ER) and are transported to the INM throughout interphase using an unknown trafficking mechanism. To study this transport, we developed a live cell assay that measures the movement of transmembrane reporters from the ER to the INM by rapamycin-mediated trapping at the nuclear lamina. Reporter constructs with small (<30 kD) cytosolic and lumenal domains rapidly accumulated at the INM. However, increasing the size of either domain by 47 kD strongly inhibited movement. Reduced temperature and ATP depletion also inhibited movement, which is characteristic of membrane fusion mechanisms, but pharmacological inhibition of vesicular trafficking had no effect. Because reporter accumulation at the INM was inhibited by antibodies to the nuclear pore membrane protein gp210, our results support a model wherein transport of integral proteins to the INM involves lateral diffusion in the lipid bilayer around the nuclear pore membrane, coupled with active restructuring of the nuclear pore complex

Dominant gain-of-function mutations in Hsp104p reveal crucial roles for the middle region

Heat-shock protein 104 (Hsp104p) is a protein-remodeling factor that promotes survival after extreme stress by disassembling aggregated proteins and can either promote or prevent the propagation of prions (protein-based genetic elements). Hsp104p can be greatly overexpressed without slowing growth, suggesting tight control of its powerful protein-remodeling activities. We isolated point mutations in Hsp104p that interfere with this control and block cell growth. Each mutant contained alterations in the middle region (MR). Each of the three MR point mutations analyzed in detail had distinct phenotypes. In combination with nucleotide binding site mutations, Hsp104p(T499I) altered bud morphology and caused septin mislocalization, colocalizing with the misplaced septins. Point mutations in the septin Cdc12p suppressed this phenotype, suggesting that it is due to direct Hsp104p–septin interactions. Hsp104p(A503V) did not perturb morphology but stopped cell growth. Remarkably, when expressed transiently, the mutant protein promoted survival after extreme stress as effectively as did wild-type Hsp104p. Hsp104p(A509D) had no deleterious effects on growth or morphology but had a greatly reduced ability to promote thermotolerance. That mutations in an 11-amino acid stretch of the MR have such profound and diverse effects suggests the MR plays a central role in regulating Hsp104p function

Degrees of controllability for quantum systems and applications to atomic systems

Precise definitions for different degrees of controllability for quantum systems are given, and necessary and sufficient conditions are discussed. The results are applied to determine the degree of controllability for various atomic systems with degenerate energy levels and transition frequencies.Comment: 20 pages, IoP LaTeX, revised and expanded versio

Quantum System Identification by Bayesian Analysis of Noisy Data: Beyond Hamiltonian Tomography

We consider how to characterize the dynamics of a quantum system from a restricted set of initial states and measurements using Bayesian analysis. Previous work has shown that Hamiltonian systems can be well estimated from analysis of noisy data. Here we show how to generalize this approach to systems with moderate dephasing in the eigenbasis of the Hamiltonian. We illustrate the process for a range of three-level quantum systems. The results suggest that the Bayesian estimation of the frequencies and dephasing rates is generally highly accurate and the main source of errors are errors in the reconstructed Hamiltonian basis.Comment: 6 pages, 3 figure

Clinical and functional characterisation of a novel TNFRSF1A c.605T > A/V173D cleavage site mutation associated with tumour necrosis factor receptor-associated periodic fever syndrome (TRAPS), cardiovascular complications and excellent response to etanercept treatment.

Objectives: To study the clinical outcome, treatment response, T-cell subsets and functional consequences of a novel tumour necrosis factor (TNF) receptor type 1 (TNFRSF1A) mutation affecting the receptor cleavage site. Methods: Patients with symptoms suggestive of tumour necrosis factor receptor-associated periodic syndrome (TRAPS) and 22 healthy controls (HC) were screened for mutations in the TNFRSF1A gene. Soluble TNFRSF1A and inflammatory cytokines were measured by ELISAs. TNFRSF1A shedding was examined by stimulation of peripheral blood mononuclear cells (PBMCs) with phorbol 12-myristate 13-acetate followed by flow cytometric analysis (FACS). Apoptosis of PBMCs was studied by stimulation with TNFa in the presence of cycloheximide and annexin V staining. T cell phenotypes were monitored by FACS. Results: TNFRSF1A sequencing disclosed a novel V173D/ p.Val202Asp substitution encoded by exon 6 in one family, the c.194–14G.A splice variant in another and the R92Q/p.Arg121Gln substitution in two families. Cardiovascular complications (lethal heart attack and peripheral arterial thrombosis) developed in two V173D patients. Subsequent etanercept treatment of the V173D carriers was highly effective over an 18-month follow-up period. Serum TNFRSF1A levels did not differ between TRAPS patients and HC, while TNFRSF1A cleavage from monocytes was significantly reduced in V173D and R92Q patients. TNFa-induced apoptosis of PBMCs and T-cell senescence were comparable between V173D patients and HC. Conclusions: The TNFRSF1A V173D cleavage site mutation may be associated with an increased risk for cardiovascular complications and shows a strong response to etanercept. T-cell senescence does not seem to have a pathogenetic role in affected patients

Organellar proteomics: the prizes and pitfalls of opening the nuclear envelope

Proteomic studies have the potential to comprehensively define the composition of organelles but are limited by the organellar cross-contamination that arises during subcellular fractionation. Comparative proteomics of organellar subfractions can mitigate these problems, as demonstrated by a recent study involving the nuclear envelope

Characterization of spatial heterogeneity of groundwater-stream water interactions using multiple depth streambed temperature measurements at the reach scale

International audienceStreambed temperatures can be easily, accurately and inexpensively measured at many locations. We obtained 140 vertical streambed temperature profiles along a 220 m section of a small artificial stream to characterize patterns of groundwater-stream water interaction with a high spatial resolution. Groundwater temperature at a sufficient depth remains nearly constant while stream water temperatures vary seasonally and diurnally. In summer, streambed temperatures of groundwater discharge zones are relatively colder than downwelling zones of stream water. Assuming vertical flow in the streambed, the observed temperatures can be correlated to the magnitude of water fluxes. The water fluxes can then be estimated by applying a simple analytical solution of the heat diffusion-advection equation to the observed vertical temperature profiles. The calculated water fluxes through the streambed ranged between 10.0 Lm?2 d?1 of stream water entering the streambed and 455.0 Lm?2 d?1 of groundwater discharging to the stream. The investigated reach was dominated by groundwater discharge with two distinct high discharge locations accounting for 50% of the total flux on 20% of the reach length