The eukaryotic plasma membrane is compartmentalized into domains enriched in specific lipids and proteins. However, our understanding of the molecular bases and biological roles of this partitioning remains incomplete. The best-studied domain in yeast is the membrane compartment containing the arginine permease Can1 (MCC) and later found to cluster additional transporters. MCCs correspond to static, furrow-like invaginations of the plasma membrane and associate with subcortical structures named "eisosomes" that include upstream regulators of the target of rapamycin complex 2 (TORC2) in the sensing of sphingolipids and membrane stress. However, how and why Can1 and other nutrient transporters preferentially segregate in MCCs remains unknown. In this study we report that the clustering of Can1 in MCCs is dictated by its conformation, requires proper sphingolipid biosynthesis, and controls its ubiquitin-dependent endocytosis. In the substrate-free outward-open conformation, Can1 accumulates in MCCs in a manner dependent on sustained biogenesis of complex sphingolipids. An arginine transport-elicited shift to an inward-facing conformation promotes its cell-surface dissipation and makes it accessible to the ubiquitylation machinery triggering its endocytosis. We further show that under starvation conditions MCCs increase in number and size, this being dependent on the BAR domain-containing Lsp1 eisosome component. This expansion of MCCs provides protection for nutrient transporters from bulk endocytosis occurring in parallel with autophagy upon TORC1 inhibition. Our study reveals nutrient-regulated protection from endocytosis as an important role for protein partitioning into membrane domains.

Serine-arginine (SR) proteins commonly designate a family of eukaryotic RNA binding proteins containing a protein domain composed of several repeats of the arginine-serine dipeptide, termed the arginine-serine (RS) domain. This protein family is involved in essential nuclear processes such as constitutive and alternative splicing of mRNA precursors. Besides participating in crucial activities in the nuclear compartment, several SR proteins are able to shuttle between the nucleus and the cytoplasm and to exert regulatory functions in the latter compartment. This review aims at discussing the properties of shuttling SR proteins with particular emphasis on their nucleo-cytoplasmic traffic and their cytoplasmic functions. Indeed, recent findings have unravelled the complex regulation of SR protein nucleo-cytoplasmic distribution and the diversity of cytoplasmic mechanisms in which these proteins are involved. SR proteins: definition and general featuresSerine-arginine (SR) proteins were first described as a family of co-purifying proteins capable of restoring splicing in S100 splicing-deficient extracts. These factors are structurally highly related as they are composed of a carboxy-terminal domain enriched with the arginine-serine (RS) dipeptide, preceded by at least one RNA binding domain of the RNA recognition motif (RRM) type [1]. These proteins play an essential role in constitutive and alternative splicing of mRNA precursors [2]. In the last 10 years, however, other functions have been unravelled for these proteins. Indeed, SR protein prototypes such as SRSF1 (ASF ⁄ SF2) and SRSF2 (SC35) were reported to stimulate transcriptional elongation (for a review see [3]). Certain SR proteins participate in mRNA translation (see [4] and below). Finally, a recent study indicates that SRSF1 promotes microRNA processing by facilitating Drosha-mediated cleavage [5]. Altogether, these findings highlight the broader roles of SR proteins in gene expression.Genome-wide searches for proteins containing RS domains revealed several other 'non-classical' SR proteins, termed SR-like proteins because of differences in the structure of the RS domain and ⁄ or lack of a prototypical RRM. While a large subset of SR-like proteins is involved in pre-mRNA processing, several members of this protein family are associated with other cellular functions [4,6].'Classical' SR proteins exist in plants [7], metazoans [1] and in some unicellular eukaryotes, such as the fission yeast Schizosaccharomyces pombe [8,9]. However, they are not present in all eukaryotes and are Abbreviations ARE, AU-rich element; NMD, nonsense-mediated decay; PIAS1, protein inhibitor of activated STAT-1; RRM, RNA recognition motif;

Edited by Ulrike KutayKeywords: Transportin Importins Karyopherins Nuclear import Nucleo-cytoplasmic transport NLS Kap104p Transportin-1 Transportin-2 Karyopherin-b2 hnRNP A1 FUS a b s t r a c t Nearly 20 years after its identification as a new b-karyopherin mediating the nuclear import of the RNA-binding protein hnRNP A1, Transportin-1 is still commonly overlooked in comparison with its best known cousin, Importin-b. Transportin-1 is nonetheless a considerable player in nucleo-cytoplasmic transport. Over the past few years, significant progress has been made in the characterization of the nuclear localization signals (NLSs) that Transportin-1 recognizes, thereby providing the molecular basis of its diversified repertoire of cargoes. The recent discovery that mutations in the Transportin-dependent NLS of FUS cause mislocalization of this protein and result in amyotrophic lateral sclerosis illustrates the importance of Transportin-dependent import for human health. Besides, new functions of Transportin-1 are emerging in processes other than nuclear import. Here, we summarize what is known about Transportin-1 and the related b-karyopherin Transportin-2. Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Basis of protein nucleo-cytoplasmic transportActive nucleo-cytoplasmic transport of proteins is mostly carried out by b-karyopherins, a family of factors functionally divided into importins and exportins. Importins bind to the nuclear localization signal (NLS) of their cargoes in the cytoplasm, either directly or through an adaptor. The importin/cargo complexes cross the nuclear pore complex (NPC) through the interactions of the importin with nucleoporins. In the nucleus, importins are bound by Ran-GTP, which releases the cargo. Importins are then recycled to the cytoplasm in association with Ran-GTP. On the cytoplasmic face of the NPC, Ran hydrolyzes its bound GTP into GDP and dissociates, freeing the importin for a new import cycle (see for example [1] for review). Exportins work in a similar way but in reverse. In the nucleus, exportins cooperatively bind Ran-GTP and a cargo featuring a fitting nuclear export signal (NES). Once the trimeric complexes reach the cytoplasm, they are dissociated and free exportins return to the nucleus to complete the cycle (see for example [2] for review). Thus, while Ran-GTP binding causes importins to release their cargoes, it is required for exportins to bind theirs. Neither importins nor exportins have significant binding affinity for the GDP-bound form of Ran. Therefore, the directionality of the transfers is maintained by mechanisms that ensure that nuclear Ran is bound to GTP and cytoplasmic Ran to GDP. The nuclear part of this task is carried out by the chromatin-associated guanine exchange factor RCC1, which promotes the exchange of GDP for GTP on nuclear Ran. Three factors located on the cytosolic face of the NPC (RanBP1, RanBP2, and RanGAP) collaborate to ensure the hydrolysis of Ran-bound GTP by Ran as soon as it goes ou...

SummaryPat1 RNA-binding proteins, enriched in processing bodies (P bodies), are key players in cytoplasmic 5′ to 3′ mRNA decay, activating decapping of mRNA in complex with the Lsm1-7 heptamer. Using co-immunoprecipitation and immunofluorescence approaches coupled with RNAi, we provide evidence for a nuclear complex of Pat1b with the Lsm2-8 heptamer, which binds to the spliceosomal U6 small nuclear RNA (snRNA). Furthermore, we establish the set of interactions connecting Pat1b/Lsm2-8/U6 snRNA/SART3 and additional U4/U6.U5 tri-small nuclear ribonucleoprotein particle (tri-snRNP) components in Cajal bodies, the site of snRNP biogenesis. RNA sequencing following Pat1b depletion revealed the preferential upregulation of mRNAs normally found in P bodies and enriched in 3′ UTR AU-rich elements. Changes in >180 alternative splicing events were also observed, characterized by skipping of regulated exons with weak donor sites. Our data demonstrate the dual role of a decapping enhancer in pre-mRNA processing as well as in mRNA decay via distinct nuclear and cytoplasmic Lsm complexes.

Newborns are characterized by poor responses to vaccines. Defective B cell responses and a Th2-type polarization can account for this impaired protection in early life. We in this study investigated the generation of follicular Th (TFH) cells, involved in the development of Ab response and germinal center reaction, upon vaccination in neonates. We showed that, compared with adults, Ab production, affinity maturation, and germinal center formation were reduced in neonates immunized with OVA–aluminum hydroxide. Although this vaccination induced CD4+ CXCR5+ PD-1+ TFH cells in newborns, their frequency, as well as their Bcl6 expression and IL-21 and IL-4 mRNA induction, was decreased in early life. Moreover, neonatal TFH cells were mainly localized in interfollicular regions of lymphoid tissues. The prototypic Th2 cytokine IL-4 was found to promote the emergence and the localization in germinal centers of neonatal TFH cells, as well as the neonatal germinal center reaction itself. In addition, IL-4 dampened expression of Th17-related molecules in neonatal TFH cells, as TFH cells from immunized IL-4–deficient neonates displayed enhanced expression of RORγt and IL-17. This Th17-like profile correlated with an increased secretion of OVA-specific IgG2a. Our study thus suggests that defective humoral immunity in early life is associated with limited and IL-4–modulated TFH cell responses.