Unidirectional Reconstitution into Detergent-destabilized Liposomes of the Purified Lactose Transport System of Streptococcus thermophilus

The lactose transport protein (LacS) of Streptococcus thermophilus was amplified to levels as high as 8 and 30% of total membrane protein in Escherichia coli and S. thermophilus, respectively. In both organisms the protein was functional and the expression levels were highest with the streptococcal lacS promoter. Also a LacS deletion mutant, lacking the carboxyl-terminal regulatory domain, could be amplified to levels >20% of membrane protein. Membranes from S. thermophilus proved to be superior in terms of efficient solubilization and ease and extent of purification of LacS; >95% of LacS was solubilized with relatively low concentrations of Triton X-100, n-octyl-β-D-glucoside, n-dodecyl-β-D-maltoside, or C12E8. The LacS protein carrying a poly-histidine tag was purified in large quantities (~5 mg/liter of culture) and with a purity >98% in a two-step process involving nickel chelate affinity and anion exchange chromatography. The membrane reconstitution of LacS was studied systematically by stepwise solubilization of preformed liposomes, prepared from E. coli phospholipid and phosphatidylcholine, and protein incorporation at the different stages of liposome solubilization. The detergents were removed by adsorption onto polystyrene beads and H+-lactose symport and lactose counterflow were measured. Highest transport activities were obtained when Triton X-100 was used throughout the solubilization/purification procedure, whereas activity was lost irreversibly with n-octyl-β-D-glucoside. For reconstitutions mediated by n-dodecyl-β-D-maltoside, C12E8, and to a lesser extent Triton X-100, the highest transport activities were obtained when the liposomes were titrated with low amounts of detergent (onset of liposome solubilization). Importantly, under these conditions proteoliposomes were obtained in which LacS was reconstituted in an inside-out orientation, as suggested by the outside labeling of a single cysteine mutant with a membrane impermeable biotin-maleimide. The results are consistent with a mechanism of reconstitution in which the hydrophilic regions of LacS prevent a random insertion of the protein into the membrane. Consistent with the in vivo lactose/galactose exchange catalyzed by the LacS protein, the maximal rate of lactose counterflow was almost 2 orders of magnitude higher than that of H+-lactose symport.

Measuring and Interpreting Nuclear Transport in Neurodegenerative Disease-The Example of C9orf72 ALS

Transport from and into the nucleus is essential to all eukaryotic life and occurs through the nuclear pore complex (NPC). There are a multitude of data supporting a role for nuclear transport in neurodegenerative diseases, but actual transport assays in disease models have provided diverse outcomes. In this review, we summarize how nuclear transport works, which transport assays are available, and what matters complicate the interpretation of their results. Taking a specific type of ALS caused by mutations in C9orf72 as an example, we illustrate these complications, and discuss how the current data do not firmly answer whether the kinetics of nucleocytoplasmic transport are altered. Answering this open question has far-reaching implications, because a positive answer would imply that widespread mislocalization of proteins occurs, far beyond the reported mislocalization of transport reporters, and specific proteins such as FUS, or TDP43, and thus presents a challenge for future research

Traffic to the inner membrane of the nuclear envelope

Past research has yielded valuable insight into the mechanisms that regulate the nuclear transport of soluble molecules like transcription factors and mRNA. Much less is known about the mechanisms responsible for the transportation of membrane proteins to the inner membrane of the nuclear envelope. The key question is: does the facilitated transport of integral inner membrane proteins exist in the same way as it does for soluble proteins and, if so, what is it used for? Herein, we provide an overview of the current knowledge on traffic to the inner nuclear membrane, and make a case that: (a) known sorting signals and molecular mechanisms in membrane protein biogenesis, membrane protein traffic and nuclear transport are also relevant with respect to INM traffic; and (b) the interplay of the effects of these signals and molecular mechanisms is what determines the rates of traffic to the INM

Evidence for the hallmarks of human aging in replicatively aging yeast

Recently, efforts have been made to characterize the hallmarks that accompany and contribute to the phenomenon of aging, as most relevant for humans [1]. Remarkably, studying the finite lifespan of the single cell eukaryote budding yeast (recently reviewed in [2] and [3]) has been paramount for our understanding of aging. Here, we compile observations from literature over the past decades of research on replicatively aging yeast to highlight how the hallmarks of aging in humans are present in yeast. We find strong evidence for the majority of these, and summarize how yeast aging is especially characterized by the hallmarks of genomic instability, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, and mitochondrial dysfunction

A FRET-based method for monitoring structural transitions in protein self-organization

Proteins assemble into a variety of dynamic and functional structures. Their structural transitions are often challenging to distinguish inside cells, particularly with a high spatiotemporal resolution. Here, we present a fluorescence resonance energy transfer (FRET)-based method for continuous and high-throughput monitoring of protein self-assemblies to reveal well-resolved transient intermediate states. Intermolecular FRET with both the donor and acceptor proteins at the same target protein provides high sensitivity while retaining the advantage of straightforward ratiometric imaging. We apply this method to monitor self-assembly of three proteins. We show that the mutant Huntingtin exon1 (mHttex1) first forms less-ordered assemblies, which develop into fibril-like aggregates, and demonstrate that the chaperone protein DNAJB6b increases the critical saturation concentration of mHttex1. We also monitor the structural changes in fused in sarcoma (FUS) condensates. This method adds to the toolbox for protein self-assembly structure and kinetics determination, and implementation with native or non-native proteins can inform studies involving protein condensation or aggregation

A simple microfluidic platform to study age-dependent protein abundance and localization changes in Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae divides asymmetrically, with a smaller daughter cell emerging from its larger mother cell. While the daughter lineage is immortal, mother cells age with each cell division and have a finite lifespan. The replicative ageing of the yeast mother cell has been used as a model to study the ageing of mitotically active human cells. Several microfluidic platforms, which use fluid flow to selectively remove daughter cells, have recently been developed that can monitor cell physiology as mother cells age. However, these platforms are not trivial to set up and users often require many hours of training. In this study, we have developed a simple system, which combines a commercially available microfluidic platform (the CellASIC ONIX Microfluidic Platform) and a genetic tool to prevent the proliferation of daughter cells (the Mother Enrichment Program), to monitor protein abundance and localization changes during approximately the first half of the yeast replicative lifespan. We validated our system by observing known age-dependent changes, such as decreased Sir2 abundance, and have identified a protein with a previously unknown age-dependent change in localization

Probing the Disordered Domain of the Nuclear Pore Complex through Coarse-Grained Molecular Dynamics Simulations

AbstractThe distribution of disordered proteins (FG-nups) that line the transport channel of the nuclear pore complex (NPC) is investigated by means of coarse-grained molecular dynamics simulations. A one-bead-per-amino-acid model is presented that accounts for the hydrophobic/hydrophilic and electrostatic interactions between different amino acids, polarity of the solvent, and screening of free ions. The results indicate that the interaction of the FG-nups forms a high-density, doughnut-like distribution inside the NPC, which is rich in FG-repeats. We show that the obtained distribution is encoded in the amino-acid sequence of the FG-nups and is driven by both electrostatic and hydrophobic interactions. To explore the relation between structure and function, we have systematically removed different combinations of FG-nups from the pore to simulate inviable and viable NPCs that were previously studied experimentally. The obtained density distributions show that the maximum density of the FG-nups inside the pore does not exceed 185 mg/mL in the inviable NPCs, whereas for the wild-type and viable NPCs, this value increases to 300 mg/mL. Interestingly, this maximum density is not correlated to the total mass of the FG-nups, but depends sensitively on the specific combination of essential Nups located in the central plane of the NPC

Size-dependent leak of soluble and membrane proteins through the yeast nuclear pore complex

Nuclear pore complexes (NPCs) allow selective import and export while forming a barrier for untargeted proteins. Using fluorescence microscopy, we measured in vivo the permeability of the Saccharomyces cerevisiae NPC for multidomain proteins of different sizes and found that soluble proteins of 150 kDa and membrane proteins with an extralumenal domain of 90 kDa were still partly localized in the nucleus on a time scale of hours. The NPCs thus form only a weak barrier for the majority of yeast proteins, given their monomeric size. Using FG triangle-mutant strains, we showed that specific combinations of Nups, especially with Nup100, but not the total mass of FG-nups per pore, were important for forming the barrier. Models of the disordered phase of wild-type and mutant NPCs were generated using a one bead per amino acid molecular dynamics model. The permeability measurements correlated with the density predictions from coarse-grained molecular dynamics simulations in the center of the NPC. The combined in vivo and computational approach provides a framework for elucidating the structural and functional properties of the permeability barrier of nuclear pore complexes

Distinctive Properties of the Nuclear Localization Signals of Inner Nuclear Membrane Proteins Heh1 and Heh2

SummaryTargeting of ER-synthesized membrane proteins to the inner nuclear membrane (INM) has long been explained by the diffusion-retention model. However, several INM proteins contain non-classical nuclear localization signal (NLS) sequences, which, in a few instances, have been shown to promote importin α/β- and Ran-dependent translocation to the INM. Here, using structural and biochemical methods, we show that yeast INM proteins Heh2 and Src1/Heh1 contain bipartite import sequences that associate intimately with the minor NLS-binding pocket of yeast importin α and unlike classical NLSs efficiently displace the IBB domain in the absence of importin β. In vivo, the intimate interactions at the minor NLS-binding pocket make the h2NLS highly efficient at recruiting importin α at the ER and drive INM localization of endogenous Heh2. Thus, h1/h2NLSs delineate a novel class of super-potent, IBB-like membrane protein NLSs, distinct from classical NLSs found in soluble cargos and of general interest in biology

A genome-wide screen identifies genes that suppress the accumulation of spontaneous mutations in young and aged yeast cells

To ensure proper transmission of genetic information, cells need to preserve and faithfully replicate their genome, and failure to do so leads to genome instability, a hallmark of both cancer and aging. Defects in genes involved in guarding genome stability cause several human progeroid syndromes, and an age-dependent accumulation of mutations has been observed in different organisms, from yeast to mammals. However, it is unclear whether the spontaneous mutation rate changes during aging and whether specific pathways are important for genome maintenance in old cells. We developed a high-throughput replica-pinning approach to screen for genes important to suppress the accumulation of spontaneous mutations during yeast replicative aging. We found 13 known mutation suppression genes, and 31 genes that had no previous link to spontaneous mutagenesis, and all acted independently of age. Importantly, we identified PEX19, encoding an evolutionarily conserved peroxisome biogenesis factor, as an age-specific mutation suppression gene. While wild-type and pex19Δ young cells have similar spontaneous mutation rates, aged cells lacking PEX19 display an elevated mutation rate. This finding suggests that functional peroxisomes may be important to preserve genome integrity specifically in old cells