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.

Age-dependent deterioration of nuclear pore assembly in mitotic cells decreases transport dynamics.

Nuclear transport is facilitated by the Nuclear Pore Complex (NPC) and is essential for life in eukaryotes. The NPC is a long-lived and exceptionally large structure. We asked whether NPC quality control is compromised in aging mitotic cells. Our images of single yeast cells during aging, show that the abundance of several NPC components and NPC assembly factors decreases. Additionally, the single-cell life histories reveal that cells that better maintain those components are longer lived. The presence of herniations at the nuclear envelope of aged cells suggests that misassembled NPCs are accumulated in aged cells. Aged cells show decreased dynamics of transcription factor shuttling and increased nuclear compartmentalization. These functional changes are likely caused by the presence of misassembled NPCs, as we find that two NPC assembly mutants show similar transport phenotypes as aged cells. We conclude that NPC interphase assembly is a major challenge for aging mitotic cells.

Active Nuclear Import of Membrane Proteins Revisited.

It is poorly understood how membrane proteins destined for the inner nuclear membrane pass the crowded environment of the Nuclear Pore Complex (NPC). For the Saccharomyces cerevisiae proteins Src1/Heh1 and Heh2, a transport mechanism was proposed where the transmembrane domains diffuse through the membrane while the extralumenal domains encoding a nuclear localization signal (NLS) and intrinsically disordered linker (L) are accompanied by transport factors and travel through the NPC. Here, we validate the proposed mechanism and explore and discuss alternative interpretations of the data. First, to disprove an interpretation where the membrane proteins become membrane embedded only after nuclear import, we present biochemical and localization data to support that the previously used, as well as newly designed reporter proteins are membrane-embedded irrespective of the presence of the sorting signals, the specific transmembrane domain (multipass or tail anchored), independent of GET, and also under conditions that the proteins are trapped in the NPC. Second, using the recently established size limit for passive diffusion of membrane proteins in yeast, and using an improved assay, we confirm active import of polytopic membrane protein with extralumenal soluble domains larger than those that can pass by diffusion on similar timescales. This reinforces that NLS-L dependent active transport is distinct from passive diffusion. Thirdly, we revisit the proposed route through the center of the NPC and conclude that the previously used trapping assay is, unfortunately, poorly suited to address the route through the NPC, and the route thus remains unresolved. Apart from the uncertainty about the route through the NPC, the data confirm active, transport factor dependent, nuclear transport of membrane-embedded mono- and polytopic membrane proteins in baker's yeast.

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 FGΔ-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.

A generic, whole-cell-based screening method for Baeyer-Villiger monooxygenases.

Baeyer-Villiger monooxygenases (BVMOs) have been receiving increasing attention as enzymes useful for biocatalytic applications. Industrial requirements call for rapid and extensive redesign of these enzymes. In response to the need for screening large libraries of BVMO mutants, we established a generic screening method that allows screening of Escherichia coli cells expressing active BVMOs in 96-well plate format. For this, we first developed an expression system for production of phenylacetone monooxygenase (PAMO) in the periplasm of E. coli. This allows probing the enzyme for any target substrate while it is also compatible with extracellular coenzyme regeneration. For coenzyme regeneration, we used phosphite dehydrogenase, which forms phosphate upon NADPH recycling. This allowed the use of a chromogenic molybdate-based phosphate determination assay. The screening procedure was supplemented with a detection method for identification of mutant enzymes that act as NADPH oxidases, thereby excluding false-positives. The whole-cell-based screening method was validated by screening site-saturation libraries of PAMO and resulted in the identification of PAMO mutants with altered catalytic properties. This new method can be used for screening libraries of BVMOs for activity with any desired substrate and therefore is a powerful tool for engineering of these enzymes.