Basic considerations to minimize bias in collection and analysis of volatile methyl siloxanes in environmental samples.

Monitoring studies that aim to quantify volatile methyl siloxanes (VMS) in environmental matrices may encounter a multitude of issues, most of which relate to the unique combination of physical-chemical characteristics of VMS that distinguish them from other classes of organic compounds. These properties, which are critical to their function in various applications, also control their fate and distribution in the environment, as well as the analytical chemistry of their measurement. Polycondensation and rearrangement reactions of VMS oligomers are possible during sample storage and analysis. Thus, care should be exercised to suppress these types of reactions by avoiding any catalytic substances or surfaces in sample collection and analysis equipment. Another factor complicating sample integrity in the analysis of trace levels of VMS, is their ubiquitous presence in many common products and components of instrumentation in the laboratory. For example, some gas chromatography columns and inlet septa have been identified as sources of VMS due to surface-catalyzed transformation of silicones to VMS promoted by moisture under high temperature in some silicone-based GC columns. Possible chemical transformation of the analytes, contamination from other sources, and potential loss of analytes need to be assessed throughout all aspects of the study, from sample collection through analysis, by establishing a rigorous quality assurance and quality control program. The implementation of such a robust QA/QC program facilitates the identification and minimization of potential analytical biases and ensures the validity and usability of data generated from environmental monitoring campaigns for VMS. The objective of this paper is to focus on aspects of collection, processing, and analysis of environmental samples that may influence the quality of the VMS analytical results. This information should then be employed in the design and implementation of future monitoring studies and can used to assess the validity of analytical results from VMS monitoring studies.

Structural and functional studies of Nup107/Nup133 interaction and its implications for the architecture of the nuclear pore complex.

Nuclear pore complexes (NPCs) are 40-60 MDa protein assemblies embedded in the nuclear envelope of eukaryotic cells. NPCs exclusively mediate all transport between cytoplasm and nucleus. The nucleoporins that build the NPC are arranged in a stable core of module-like subcomplexes with eight-fold rotational symmetry. To gain insight into the intricate assembly of the NPC, we have solved the crystal structure of a protein complex between two nucleoporins, human Nup107 and Nup133. Both proteins form elongated structures that interact tightly via a compact interface in tail-to-tail fashion. Additional experiments using structure-guided mutants show that Nup107 is the critical anchor for Nup133 to the NPC, positioning Nup133 at the periphery of the NPC. The significant topological differences between Nup107 and Nup133 suggest that *-helical nucleoporin domains of the NPC scaffold fall in different classes and fulfill largely nonredundant functions.

Purification, crystallization and preliminary X-ray analysis of a Nup107-Nup133 heterodimeric nucleoporin complex.

The nuclear pore complex (NPC), the sole gateway of traffic between the nucleus and the cytoplasm, is built up from multiple copies of about 30 proteins collectively termed nucleoporins (nups). Nups are organized into distinct subcomplexes. Nup107 and Nup133 are members of the essential Nup107-160 subcomplex, a component of the central NPC architecture. A dimeric complex of the C-terminal domains of human Nup107 and Nup133 was expressed from a bicistronic vector in Escherichia coli, purified and crystallized in two different crystal forms. Crystals grown in the presence of 18-22% PEG 3350 belong to space group P2(1)2(1)2(1) and diffracted to 2.9 A. Native and seleno-L-methionine-derivative crystals grown in the presence of 1.1 M sodium malonate belong to space group C2 and diffracted to 2.55 and 2.9 A, respectively. Structure determination of this complex will give the first insights into the protein-protein interactions within a core module of the NPC.

Cell-cycle-dependent phosphorylation of the nuclear pore Nup107-160 subcomplex.

The nuclear pore complex (NPC) mediates macromolecular transport between the nucleus and the cytoplasm. Many NPC proteins (nucleoporins, Nups) are modified by phosphorylation. It is believed that phosphorylation regulates the breakdown of the nuclear envelope at mitosis and the disassembly of the NPC into different subcomplexes. In this study, we examined the cell-cycle-dependent phosphorylation of the Nup107-160 subcomplex, a core building block of the NPC. Using in vivo (32)P labeling in HeLa cells, we found that Nup107, Nup96, and Nup133 are phosphorylated during mitosis. To precisely map the phosphorylation sites within the complex, we used a comprehensive multiple-stage MS approach (MS, MS(2), and MS(3)), establishing that Nup160, Nup133, Nup96, and Nup107 are all targets of phosphorylation. We determined that the phosphorylation sites are clustered mainly at the N-terminal regions of these proteins, which are predicted to be natively disordered. In addition, we determined the cell-cycle dependence of the phosphorylation of these sites by using stable isotope labeling and MS(2) analysis. Measurement of the site-specific phosphorylation ratios between mitotic and G(1) cells led us to conclude that several phosphorylation events of the subcomplex are mainly mitotic. Based on these results and our finding that the entire Nup107-160 subcomplex is stable throughout the cell cycle, we propose that phosphorylation does not affect interactions within the Nup107-160 subcomplex, but regulates the association of the subcomplex with the NPC and other proteins.

A general amphipathic alpha-helical motif for sensing membrane curvature.

The Golgi-associated protein ArfGAP1 has an unusual membrane-adsorbing amphipathic alpha-helix: its polar face is weakly charged, containing mainly serine and threonine residues. We show that this feature explains the specificity of ArfGAP1 for curved versus flat lipid membranes. We built an algorithm to identify other potential amphipathic alpha-helices rich in serine and threonine residues in protein databases. Among the identified sequences, we show that three act as membrane curvature sensors. In the golgin GMAP-210, the sensor may serve to trap small vesicles at the end of a long coiled coil. In Osh4p/Kes1p, which transports sterol between membranes, the sensor controls access to the sterol-binding pocket. In the nucleoporin Nup133, the sensor corresponds to an exposed loop of a beta-propeller structure. Ser/Thr-rich amphipathic helices thus define a general motif used by proteins of various functions for sensing membrane curvature.

Structural and functional analysis of Nup133 domains reveals modular building blocks of the nuclear pore complex.

Nucleocytoplasmic transport occurs through nuclear pore complexes (NPCs) whose complex architecture is generated from a set of only approximately 30 proteins, termed nucleoporins. Here, we explore the domain structure of Nup133, a nucleoporin in a conserved NPC subcomplex that is crucial for NPC biogenesis and is believed to form part of the NPC scaffold. We show that human Nup133 contains two domains: a COOH-terminal domain responsible for its interaction with its subcomplex through Nup107; and an NH2-terminal domain whose crystal structure reveals a seven-bladed beta-propeller. The surface properties and conservation of the Nup133 beta-propeller suggest it may mediate multiple interactions with other proteins. Other beta-propellers are predicted in a third of all nucleoporins. These and several other repeat-based motifs appear to be major elements of nucleoporins, indicating a level of structural repetition that may conceptually simplify the assembly and disassembly of this huge protein complex.

Depletion of a single nucleoporin, Nup107, prevents the assembly of a subset of nucleoporins into the nuclear pore complex.

The nuclear pore complex (NPC) is a protein assembly that contains several distinct subcomplexes. The mammalian nucleoporin (Nup)-107 is part of a hetero-oligomeric complex, that also contains Nup160, Nup133, Nup96, and the mammalian homolog of yeast Sec13p. We used transfection of HeLa cells with small interfering RNAs to specifically deplete mRNA for Nup107. In a domino effect, Nup107 depletion caused codepletion of a subset of other Nups on their protein but not on their mRNA level. Among the affected Nups was a member of the Nup107 subcomplex, Nup133, whereas two other tested members of this complex, Nup96 and Sec13, were unaffected and assembled into Nup107Nup133-deficient NPCs. We also tested several phenylalanine-glycine repeat-containing Nups that serve as docking sites for karyopherins. Some of these, such as Nup358, Nup214 on the cytoplasmic, and Nup153 on the nucleoplasmic side of the NPC, failed to assemble into Nup107Nup133-depleted NPCs, whereas p62, a Nup at the center of the NPC, was unaffected. Interestingly, the filamentous, NPC-associated protein Tpr also failed to assemble into the NPCs of Nup107-depleted cells. These data indicate that Nup107 functions as a keystone Nup that is required for the assembly of a subset of Nups into the NPC. Despite the depletion of Nup107 and the accompanying effects on other Nups, there was no significant effect on the growth rate of these cells and only a partial inhibition of mRNA export. These data indicate redundancy of Nups in the function of the mammalian NPC.