Lipid Droplets Embedded in a Model Cell Membrane Create a Phospholipid Diffusion Barrier.

Lipid droplets (LDs) are ubiquitous, cytoplasmic fat storage organelles that originate from the endoplasmic reticulum (ER) membrane. They are composed of a core of neutral lipids surrounded by a phospholipid monolayer. Proteins embedded into this monolayer membrane adopt a monotopic topology and are crucial for regulated lipid storage and consumption. A key question is, which collective properties of protein-intrinsic and lipid-mediated features determine spatio-temporal protein partitioning between phospholipid bilayer and LD monolayer membranes. To address this question, a freestanding phospholipid bilayer with physiological lipidic composition is produced using microfluidics and micrometer-sized LDs are dispersed around the bilayer that spontaneously insert into the bilayer. Using confocal microscopy, the 3D geometry of the reconstituted LDs is determined with high spatial resolution. The micrometer-sized bilayer-embedded LDs present a characteristic lens shape that obeys predictions from equilibrium wetting theory. Fluorescence recovery after photobleaching measurements reveals the existence of a phospholipid diffusion barrier at the monolayer-bilayer interface. Coarse-grained molecular dynamics simulation reveals lipid specific density distributions along the pore rim, which may rationalize the diffusion barrier. The lipid diffusion barrier between the LD covering monolayer and the bilayer may be a key phenomenon influencing protein partitioning between the ER membrane and LDs in living cells.

Mechanisms of protein targeting to lipid droplets: A unified cell biological and biophysical perspective.

Lipid droplets (LDs), or oil bodies in plants, are specialized organelles that primarily serve as hubs of cellular metabolic energy storage and consumption. These ubiquitous cytoplasmic organelles are derived from the endoplasmic reticulum (ER) and consist of a hydrophobic neutral lipid core - mainly consisting of triglycerides and sterol esters - that is encircled by a phospholipid monolayer. The dynamic metabolic functions of the LDs are mainly executed and regulated by proteins on the monolayer surface. However, its unique architecture puts some structural constraints on the types of proteins that can associate with LDs. The lipid monolayer is decorated with either peripheral proteins or with integral membrane proteins that adopt a monotopic topology. Due to its oil-water interface, which is energetically costly, the LD surface happens to be favorable to the recruitment of many proteins involved in metabolic but also non-metabolic functions. We only started very recently to understand biophysical and biochemical principles controlling protein targeting to LDs. This review aims to summarize the most recent findings regarding this topic and proposes directions that will potentially lead to a better understanding of LD surface characteristics, as compared to bilayer membranes, and how that impacts protein-LD interactions.

SUMOylation of the nuclear pore complex basket is involved in sensing cellular stresses.

The nuclear pore complex (NPC) is the major conduit for nucleocytoplasmic transport and serves as a platform for gene regulation and DNA repair. Several nucleoporins undergo ubiquitylation and SUMOylation, and these modifications play an important role in nuclear pore dynamics and plasticity. Here, we perform a detailed analysis of these post-translational modifications of yeast nuclear basket proteins under normal growth conditions as well as upon cellular stresses, with a focus on SUMOylation. We find that the balance between the dynamics of SUMOylation and deSUMOylation of Nup60 and Nup2 at the NPC differs substantially, particularly in G1 and S phase. While Nup60 is the unique target of genotoxic stress within the nuclear basket that probably belongs to the SUMO-mediated DNA damage response pathway, both Nup2 and Nup60 show a dramatic increase in SUMOylation upon osmotic stress, with Nup2 SUMOylation being enhanced in Nup60 SUMO-deficient mutant yeast strains. Taken together, our data reveal that there are several levels of crosstalk between nucleoporins, and that the post-translational modifications of the NPC serve in sensing cellular stress signals.

A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly.

Within a single generation time a growing yeast cell imports ∼14 million ribosomal proteins (r-proteins) into the nucleus for ribosome production. After import, it is unclear how these intrinsically unstable and aggregation-prone proteins are targeted to the ribosome assembly site in the nucleolus. Here, we report the discovery of a conserved nuclear carrier Tsr2 that coordinates transfer of the r-protein eS26 to the earliest assembling pre-ribosome, the 90S. In vitro studies revealed that Tsr2 efficiently dissociates importin:eS26 complexes via an atypical RanGTP-independent mechanism that terminates the import process. Subsequently, Tsr2 binds the released eS26, shields it from proteolysis, and ensures its safe delivery to the 90S pre-ribosome. We anticipate similar carriers-termed here escortins-to securely connect the nuclear import machinery with pathways that deposit r-proteins onto developing pre-ribosomal particles.

Sumoylation regulates Kap114-mediated nuclear transport.

The nuclear import receptor Kap114 carries transcription factors and other cargos across nuclear pores into the nucleus. Here we show that yeast Kap114 is modified by SUMO (small ubiquitin-related modifier) and that sumoylation is required for Kap114-mediated nuclear import. Among the four known SUMO-specific E3 ligases in yeast, Mms21 is the preferred E3 enzyme responsible for the covalent attachment of SUMO to the Kap114 protein. Kap114 is sumoylated on lysine residue 909, which is part of a ΨKxD/E sumoylation consensus motif. Kap114 containing a lysine-to-arginine point mutation at position 909 mislocalizes to the nucleus and is defective in promoting nuclear import. Similarly, mutants defective in sumoylation or desumoylation specifically accumulate Kap114 in the nucleus and are blocked in import of Kap114 cargos. Ran-GTP is not sufficient to disassemble Kap114/cargo complexes, which necessitates additional cargo release mechanisms in the nucleus. Remarkably, sumoylation of Kap114 greatly stimulates cargo dissociation in vitro. We propose that sumoylation occurs at the site of Kap114 cargo function and that SUMO is a cargo release factor involved in intranuclear targeting.

Yeast Mpk1 cell wall integrity mitogen-activated protein kinase regulates nucleocytoplasmic shuttling of the Swi6 transcriptional regulator.

The yeast SBF transcription factor is a heterodimer comprised of Swi4 and Swi6 that has a well defined role in cell cycle-specific transcription. SBF serves a second function in the transcriptional response to cell wall stress in which activated Mpk1 mitogen-activated protein kinase of the cell wall integrity signaling pathway forms a complex with Swi4, the DNA binding subunit of SBF, conferring upon Swi4 the ability to bind DNA and activate transcription of FKS2. Although Mpk1-Swi4 complex formation and transcriptional activation of FKS2 does not require Mpk1 catalytic activity, Swi6 is phosphorylated by Mpk1 and must be present in the Mpk1-Swi4 complex for transcriptional activation of FKS2. Here, we find that Mpk1 regulates Swi6 nucleocytoplasmic shuttling in a biphasic manner. First, formation of the Mpk1-Swi4 complex recruits Swi6 to the nucleus for transcriptional activation. Second, Mpk1 negatively regulates Swi6 by phosphorylation on Ser238, which inhibits nuclear entry. Ser238 neighbors a nuclear localization signal (NLS) whose function is blocked by phosphorylation at Ser238 in a manner similar to the regulation by Cdc28 of another Swi6 NLS, revealing a mechanism for the integration of multiple signals to a single endpoint. Finally, the Kap120 beta-importin binds the Mpk1-regulated Swi6 NLS but not the Cdc28-regulated NLS.

Classical NLS proteins from Saccharomyces cerevisiae.

Proteins can enter the nucleus through various receptor-mediated import pathways. One class of import cargos carries a classical nuclear localization signal (cNLS) containing a short cluster of basic residues. This pathway involves importin alpha (Impalpha), which possesses the cNLS binding site, and importin beta (Impbeta), which translocates the import complex through the nuclear pore complex. The defining criteria for a cNLS protein from Saccharomyces cerevisiae are an in vivo import defect in Impalpha and Impbeta mutants, direct binding to purified Impalpha, and stimulation of this binding by Impbeta. We show for the first time that endogenous S. cerevisiae proteins Prp20, Cdc6, Swi5, Cdc45, and Clb2 fulfill all of these criteria identifying them as authentic yeast cNLS cargos. Furthermore, we found that the targeting signal of Prp20 is a bipartite cNLS and that of Cdc6 is a monopartite cNLS. Basic residues present within these motifs are of different significance for the interaction with Impalpha. We determined the binding constants for import complexes containing the five cNLS proteins by surface plasmon resonance spectrometry. The dissociation constants for cNLS/alpha/beta complexes differ considerably, ranging from 1 nM for Cdc6 to 112 nM for Swi5, suggesting that the nuclear import kinetics is determined by the strength of cNLS/Impalpha binding. Impbeta enhances the affinity of Impalpha for cNLSs approximately 100-fold. This stimulation of cNLS binding to Impalpha results from a faster association in the presence of Impbeta, whereas the dissociation rate is unaffected by Impbeta. This implies that, after entry into the nucleus, the release of Impbeta by the Ran guanosine triphosphatase (Ran GTPase) from the import complex is not sufficient to dissociate the cNLS/Impalpha subcomplex. Our observation that the nucleoporin Nup2, which had been previously shown to release the cNLS from Impalpha in vitro, is required for efficient import of all the genuine cNLS cargos supports a general role of Nup2 in import termination.

A novel conserved nuclear localization signal is recognized by a group of yeast importins.

Nucleo-cytoplasmic transport of proteins is mostly mediated by specific interaction between transport receptors of the importin beta family and signal sequences present in their cargo. While several signal sequences, in particular the classical nuclear localization signal (NLS) recognized by the heterodimeric importin alpha/beta complex are well known, the signals recognized by other importin beta-like transport receptors remain to be characterized in detail. Here we present the systematic analysis of the nuclear import of Saccharomyces cerevisiae Asr1p, a nonessential alcohol-responsive Ring/PHD finger protein that shuttles between nucleus and cytoplasm but accumulates in the nucleus upon alcohol stress. Nuclear import of Asr1p is constitutive and mediated by its C-terminal domain. A short sequence comprising residues 243-280 is sufficient and necessary for active targeting to the nucleus. Moreover, the nuclear import signal is conserved from yeast to mammals. In vitro, the nuclear localization signal of Asr1p directly interacts with the importins Kap114p, Kap95p, Pse1p, Kap123p, or Kap104p, interactions that are sensitive to the presence of RanGTP. In vivo, these importins cooperate in nuclear import. Interestingly, the same importins mediate nuclear transport of histone H2A. Based on mutational analysis and sequence comparison with a region mediating nuclear import of histone H2A, we identified a novel type of NLS with the consensus sequence R/KxxL(x)(n)V/YxxV/IxK/RxxxK/R that is recognized by five yeast importins and connects them into a highly efficient network for nuclear import of proteins.

Kap120 functions as a nuclear import receptor for ribosome assembly factor Rpf1 in yeast.

The nucleocytoplasmic exchange of macromolecules is mediated by receptors specialized in passage through the nuclear pore complex. The majority of these receptors belong to the importin beta protein family, which has 14 members in Saccharomyces cerevisiae. Nine importins carry various cargos from the cytoplasm into the nucleus, whereas four exportins mediate nuclear export. Kap120 is the only receptor whose transport cargo has not been found previously. Here, we characterize Kap120 as an importin for the ribosome maturation factor Rpf1, which was identified in a two-hybrid screen. Kap120 binds directly to Rpf1 in vitro and is released by Ran-GTP. At least three parallel import pathways exist for Rpf1, since nuclear import is defective in strains with the importins Kap120, Kap114, and Nmd5 deleted. Both kap120 and rpf1 mutants accumulate large ribosomal subunits in the nucleus. The nuclear accumulation of 60S ribosomal subunits in kap120 mutants is abolished upon RPF1 overexpression, indicating that Kap120 does not function in the actual ribosomal export step but rather in import of ribosome maturation factors.

The histones H2A/H2B and H3/H4 are imported into the yeast nucleus by different mechanisms.

Proteins are imported from the cytoplasm into the nucleus by importin beta-related transport receptors. The yeast Saccharomyces cerevisiae contains ten of these importins, but only two of them are essential. After transfer through the nuclear pore, importins release their cargo upon binding to the Ran GTPase, the key regulator of nuclear transport. We investigated the import of the core histones in yeast and found that four importins are involved. The essential Pse1p and the nonessential importins Kap114p, Kap104p, and Yrb4p/Kap123p specifically bind to histones H2A and H2B. Release of H2 histones from importins requires Ran-GTP and DNA simultaneously suggesting a function of the importins in intranuclear targeting. H3 and H4 associate mainly with Pse1p and the dissociation requires Ran but not DNA, which points to a different import mechanism. Import of green fluorescent protein fusions to H2A and H2B requires primarily Pse1p and Kap114p, whereas Yrb4p plays an auxiliary role. Pse1p is predominantly necessary for nuclear uptake of H3 and H4, while Kap104p and Yrb4p also support import. We conclude from our in vivo and in vitro experiments that import of the essential histones is mediated mainly by the essential importin Pse1p, while the non-essential Kap114p functions in a parallel import pathway for H2A and H2B.