DNA circles promote yeast ageing in part through stimulating the reorganization of nuclear pore complexes.

The nuclear pore complex (NPC) mediates nearly all exchanges between nucleus and cytoplasm, and in many species, it changes composition as the organism ages. However, how these changes arise and whether they contribute themselves to ageing is poorly understood. We show that SAGA-dependent attachment of DNA circles to NPCs in replicatively ageing yeast cells causes NPCs to lose their nuclear basket and cytoplasmic complexes. These NPCs were not recognized as defective by the NPC quality control machinery (SINC) and not targeted by ESCRTs. They interacted normally or more effectively with protein import and export factors but specifically lost mRNA export factors. Acetylation of Nup60 drove the displacement of basket and cytoplasmic complexes from circle-bound NPCs. Mutations preventing this remodeling extended the replicative lifespan of the cells. Thus, our data suggest that the anchorage of accumulating circles locks NPCs in a specialized state and that this process is intrinsically linked to the mechanisms by which ERCs promote ageing.

Flexible and Extended Linker Domains Support Efficient Targeting of Heh2 to the Inner Nuclear Membrane.

The nuclear pore complex (NPC) is embedded in the nuclear envelope and forms the main gateway to the nuclear interior including the inner nuclear membrane (INM). Two INM proteins in yeast are selectively imported. Their sorting signals consist of a nuclear localization signal, separated from the transmembrane domain by a long intrinsically disordered (ID) linker. We used computational models to predict the dynamic conformations of ID linkers and analyzed the INM targeting efficiency of proteins with linker regions with altered Stokes radii and decreased flexibilities. We find that flexibility, Stokes radius, and the frequency at which the linkers are at an extended end-to-end distance larger than 25 nm are good predictors for the targeting of the proteins. The data are consistent with a transport mechanism in which INM targeting of Heh2 is dependent on an ID linker that facilitates the crossing of the approximately 25-nm thick NPC scaffold.

Saccharomyces cerevisiae goes through distinct metabolic phases during its replicative lifespan.

A comprehensive description of the phenotypic changes during cellular aging is key towards unraveling its causal forces. Previously, we mapped age-related changes in the proteome and transcriptome (Janssens et al., 2015). Here, employing the same experimental procedure and model-based inference, we generate a comprehensive account of metabolic changes during the replicative life of Saccharomyces cerevisiae. With age, we found decreasing metabolite levels, decreasing growth and substrate uptake rates accompanied by a switch from aerobic fermentation to respiration, with glycerol and acetate production. The identified metabolic fluxes revealed an increase in redox cofactor turnover, likely to combat increased production of reactive oxygen species. The metabolic changes are possibly a result of the age-associated decrease in surface area per cell volume. With metabolism being an important factor of the cellular phenotype, this work complements our recent mapping of the transcriptomic and proteomic changes towards a holistic description of the cellular phenotype during aging.

Protein biogenesis machinery is a driver of replicative aging in yeast.

An integrated account of the molecular changes occurring during the process of cellular aging is crucial towards understanding the underlying mechanisms. Here, using novel culturing and computational methods as well as latest analytical techniques, we mapped the proteome and transcriptome during the replicative lifespan of budding yeast. With age, we found primarily proteins involved in protein biogenesis to increase relative to their transcript levels. Exploiting the dynamic nature of our data, we reconstructed high-level directional networks, where we found the same protein biogenesis-related genes to have the strongest ability to predict the behavior of other genes in the system. We identified metabolic shifts and the loss of stoichiometry in protein complexes as being consequences of aging. We propose a model whereby the uncoupling of protein levels of biogenesis-related genes from their transcript levels is causal for the changes occurring in aging yeast. Our model explains why targeting protein synthesis, or repairing the downstream consequences, can serve as interventions in aging.

Integrated quantification and identification of aldehydes and ketones in biological samples.

The identification of unknown compounds remains to be a bottleneck of mass spectrometry (MS)-based metabolomics screening experiments. Here, we present a novel approach which facilitates the identification and quantification of analytes containing aldehyde and ketone groups in biological samples by adding chemical information to MS data. Our strategy is based on rapid autosampler-in-needle-derivatization with p-toluenesulfonylhydrazine (TSH). The resulting TSH-hydrazones are separated by ultrahigh-performance liquid chromatography (UHPLC) and detected by electrospray ionization-quadrupole-time-of-flight (ESI-QqTOF) mass spectrometry using a SWATH (Sequential Window Acquisition of all Theoretical Fragment-Ion Spectra) data-independent high-resolution mass spectrometry (HR-MS) approach. Derivatization makes small, poorly ionizable or retained analytes amenable to reversed phase chromatography and electrospray ionization in both polarities. Negatively charged TSH-hydrazone ions furthermore show a simple and predictable fragmentation pattern upon collision induced dissociation, which enables the chemo-selective screening for unknown aldehydes and ketones via a signature fragment ion (m/z 155.0172). By means of SWATH, targeted and nontargeted application scenarios of the suggested derivatization route are enabled in the frame of a single UHPLC-ESI-QqTOF-HR-MS workflow. The method's ability to simultaneously quantify and identify molecules containing aldehyde and ketone groups is demonstrated using 61 target analytes from various compound classes and a (13)C labeled yeast matrix. The identification of unknowns in biological samples is detailed using the example of indole-3-acetaldehyde.

In silico approaches and the role of ontologies in aging research.

The 2013 Rostock Symposium on Systems Biology and Bioinformatics in Aging Research was again dedicated to dissecting the aging process using in silico means. A particular focus was on ontologies, because these are a key technology to systematically integrate heterogeneous information about the aging process. Related topics were databases and data integration. Other talks tackled modeling issues and applications, the latter including talks focused on marker development and cellular stress as well as on diseases, in particular on diseases of kidney and skin.

The nuclear basket proteins Mlp1p and Mlp2p are part of a dynamic interactome including Esc1p and the proteasome.

The basket of the nuclear pore complex (NPC) is generally depicted as a discrete structure of eight protein filaments that protrude into the nucleoplasm and converge in a ring distal to the NPC. We show that the yeast proteins Mlp1p and Mlp2p are necessary components of the nuclear basket and that they also embed the NPC within a dynamic protein network, whose extended interactome includes the spindle organizer, silencing factors, the proteasome, and key components of messenger ribonucleoproteins (mRNPs). Ultrastructural observations indicate that the basket reduces chromatin crowding around the central transporter of the NPC and might function as a docking site for mRNP during nuclear export. In addition, we show that the Mlps contribute to NPC positioning, nuclear stability, and nuclear envelope morphology. Our results suggest that the Mlps are multifunctional proteins linking the nuclear transport channel to multiple macromolecular complexes involved in the regulation of gene expression and chromatin maintenance.

Quantitative analysis of membrane protein transport across the nuclear pore complex.

Nuclear transport of the Saccharomyces cerevisiae membrane proteins Src1/Heh1 and Heh2 across the NPC is facilitated by a long intrinsically disordered linker between the nuclear localization signal (NLS) and the transmembrane domain. The import of reporter proteins derived from Heh2 is dependent on the FG-Nups in the central channel, and the linker can position the transport factor-bound NLS in the vicinity of the FG-Nups in the central channel, while the transmembrane segment resides in the pore membrane. Here, we present a quantitative analysis of karyopherin-mediated import and passive efflux of reporter proteins derived from Heh2, including data on the mobility of the reporter proteins in different membrane compartments. We show that membrane proteins with extralumenal domains up to 174 kDa, terminal to the linker and NLS, passively leak out of the nucleus via the NPC, albeit at a slow rate. We propose that also during passive efflux, the unfolded linker facilitates the passage of extralumenal domains through the central channel of the NPC.

The transport of integral membrane proteins across the nuclear pore complex.

The nuclear envelope protects and organizes the genome. The nuclear pore complexes embedded in the nuclear envelope allow selective transport of macromolecules between the cytosol and nucleoplasm, and as such help to control the flow of information from DNA to RNA to proteins. A growing list of integral membrane proteins of the nuclear envelope are described to function in the organization of the genome, as well as the assembly of the NPCs. Here, we discuss how the nuclear pore complex may sort these proteins to obtain a specific protein composition of the inner membrane.

Long unfolded linkers facilitate membrane protein import through the nuclear pore complex.

Active nuclear import of soluble cargo involves transport factors that shuttle cargo through the nuclear pore complex (NPC) by binding to phenylalanine-glycine (FG) domains. How nuclear membrane proteins cross through the NPC to reach the inner membrane is presently unclear. We found that at least a 120-residue-long intrinsically disordered linker was required for the import of membrane proteins carrying a nuclear localization signal for the transport factor karyopherin-α. We propose an import mechanism for membrane proteins in which an unfolded linker slices through the NPC scaffold to enable binding between the transport factor and the FG domains in the center of the NPC.

A karyopherin acts in localized protein synthesis.

Multiple mechanisms are in place to regulate adequate synthesis of proteins, ranging from ways to ensure sequence fidelity, polypeptide folding and protein modification, to control of amounts and subcellular localization of the molecules. Some of these mechanisms act at the level of mRNA export and mRNA targeting. mRNA nuclear export consists of three coupled consecutive steps: (1) the packaging into messenger ribonucleoprotein (mRNP); (2) the transport through the nuclear pore complexes (NPCs); and (3) the directional release into the cytoplasm (reviewed in refs. 1 and 2). The subsequent targeting of mRNA to particular subcellular locations is common in asymmetric cell division in many eukaryotes (reviewed in refs. 3­5) and ensures that proteins are produced at the desired place. Recent studies in Saccharomyces cerevisiae suggest that Karyopherin Kap104p plays a role not only in mRNA export but also in bud-localized protein synthesis. In this report, we reflect on the possible mechanisms by which Kap104p links these events and hypothesize on a possible function of the localized protein synthesis.

Nuclear transport factor directs localization of protein synthesis during mitosis.

Export of messenger RNA from the transcription site in the nucleus and mRNA targeting to the translation site in the cytoplasm are key regulatory processes in protein synthesis. In yeast, the mRNA-binding proteins Nab2p and Nab4p/Hrp1p accompany transcripts to their translation site, where the karyopherin Kap104p mediates both their dissociation from the mRNA and their transport back into the nucleus. We found that Kap104p localized to the distal bud tip and the bud neck during cell division, resulting in a localized release of translation-competent mRNA and increased protein synthesis in the emerging daughter cell. Temporally and spatially coordinated localization of Kap104p is a new mechanism for the asymmetric distribution of protein synthesis in dividing cells.