Friedrich Miescher Prize awardee lecture review. A conserved family of nuclear export receptors mediates the exit of messenger RNA to the cytoplasm

The distinguishing feature of eukaryotic cells is the segregation of RNA biogenesis and DNA replication in the nucleus, separate from the cytoplasmic machinery for protein synthesis. As a consequence, messenger RNAs (mRNAs) and all cytoplasmic RNAs from nuclear origin need to be transported from their site of synthesis in the nucleus to their final cytoplasmic destination. Nuclear export occurs through nuclear pore complexes (NPCs) and is mediated by saturable transport receptors, which shuttle between the nucleus and cytoplasm. The past years have seen great progress in the characterization of the mRNA export pathway and the identification of proteins involved in this process. A novel family of nuclear export receptors (the NXF family), distinct from the well-characterized family of importin β-like proteins, has been implicated in the export of mRNA to the cytoplasm. Received 23 January 2001; received after revision 12 April 2001; accepted 12 April 2001     

The nuclear envelope: emerging roles in development and disease

The chromosomes of eukaryotic cells are separated from the cytoplasm by the nuclear envelope. The nuclear envelope includes two riveted membranes, plus embedded pore complexes that mediate nuclear import and export. In this sense, the nuclear envelope is truly a border zone. However, the envelope also links directly to chromosomes, and anchors two major infrastructures--the nuclear lamina and Tpr filaments--to the nuclear perimeter. Proteins of the nuclear envelope mediate a variety of fundamental activities, including DNA replication, gene expression and silencing, chromatin organization, cell division, apoptosis, sperm nuclear remodeling, the behavior of pronuclei, cell fate determination, nuclear migration and cell polarity. Furthermore, mutations in nuclear lamins and lamin-binding proteins cause tissue-specific inherited diseases. This special issue of Cell and Molecular Life Sciences is devoted to recent major advances in the characterization of nuclear envelope proteins and their roles. We offer here an overview of the topics covered in this issue of CMLS, and also discuss the emerging recognition that the nuclear envelope is an organelle critical for a wide range of genetic and developmental activity in multicellular organisms.     

The role of the nuclear envelope in cellular organization

Over the last years it has become evident that the nuclear envelope (NE) is more than a passive membrane barrier that separates the nucleus from the cytoplasm. The NE not only controls the trafficking of macromolecules between the nucleoplasm and the cytosol, but also provides anchoring sites for chromosomes and cytoskeleton to the nuclear periphery. Targeting of chromatin to the NE might actually be part of gene expression regulation in eukaryotes. Mutations in certain NE proteins are associated with a diversity of human diseases, including muscular dystrophy, neuropathy, lipodistrophy, torsion dystonia and the premature aging condition progeria. Despite the importance of the NE for cell division and differentiation, relatively little is known about its biogenesis and its role in human diseases. It is our goal to provide a comprehensive view of the NE and to discuss possible implications of NE-associated changes for gene expression, chromatin organization and signal transduction. Received 8 August 2005; received after revision 13 October 2005; accepted 13 October 2005     

Nuclear envelope and nuclear matrix: interactions and dynamics

The peripheral nuclear lamina is located near the nuclear inner membrane and consists of lamin filaments and integral membrane proteins, including the lamin B receptor and various isoforms of lamina-associated polypeptides (LAP) 1 and 2. Several nuclear membrane proteins also interact with chromatin proteins BAF and Hp1. Lamins in the nuclear interior associate with at least one soluble (non-membrane-bound) LAP2 isoform named LAP2alpha. The internal lamins, together with Tpr-based filaments that connect to nuclear pore complexes, are proposed to be major structural elements of the internal nuclear matrix. We describe the structural links between the peripheral lamina and the internal nuclear matrix that are thought to be mediated by LAP2 family members, filament protein Tpr and nucleoporin Nup153. These findings are discussed in relation to human diseases that arise from mutations in nuclear lamina proteins.     

Many mechanisms, one entrance: membrane protein translocation into the nucleus

The inner nuclear membrane harbors a unique set of membrane proteins, many of which interact with nuclear intermediate filaments and chromatin components and thus play an important role in nuclear organization and gene expression regulation. These membrane proteins have to be constantly transported into the nucleus from their sites of synthesis in the ER to match the growth of the nuclear membrane during interphase. Many mechanisms have evolved to enable translocation of these proteins to the nucleus. The full range of mechanisms goes from rare autophagy events to regulated translocation using the nuclear pore complexes. Though mechanisms involving nuclear pores are predominant, within this group an enormous mechanistic range is observed from free diffusion through the peripheral channels to many distinct mechanisms involving different nucleoporins and other components of the soluble protein transport machinery in the central channels. This review aims to provide a comprehensive insight into this mechanistic diversity.     

Serine residue 115 of MAPK-activated protein kinase MK5 is crucial for its PKA-regulated nuclear export and biological function

The mitogen-activated protein kinase-activated protein kinase-5 (MK5) resides predominantly in the nucleus of resting cells, but p38^MAPK, extracellular signal-regulated kinases-3 and -4 (ERK3 and ERK4), and protein kinase A (PKA) induce nucleocytoplasmic redistribution of MK5. The mechanism by which PKA causes nuclear export remains unsolved. In the study reported here we demonstrated that Ser-115 is an in vitro PKA phosphoacceptor site, and that PKA, but not p38^MAPK, ERK3 or ERK4, is unable to redistribute MK5 S115A to the cytoplasm. However, the phosphomimicking MK5 S115D mutant resides in the cytoplasm in untreated cells. While p38^MAPK, ERK3 and ERK4 fail to trigger nuclear export of the kinase dead T182A and K51E MK5 mutants, S115D/T182A and K51E/S115D mutants were able to enter the cytoplasm of resting cells. Finally, we demonstrated that mutations in Ser-115 affect the biological properties of MK5. Taken together, our results suggest that Ser-115 plays an essential role in PKA-regulated nuclear export of MK5, and that it also may regulate the biological functions of MK5.     

<Emphasis Type="Italic">Toxoplasma gondii</Emphasis> chromosomal passenger complex is essential for the organization of a functional mitotic spindle: a prerequisite for productive endodyogeny

The phylum Apicomplexa encompasses deadly pathogens such as malaria and Cryptosporidium. Apicomplexa cell division is mechanistically divergent from that of their mammalian host, potentially representing an attractive source of drug targets. Depending on the species, apicomplexan parasites can modulate the output of cell division, producing two to thousands of daughter cells at once. The inherent flexibility of their cell division mechanisms allows these parasites to adapt to different niches, facilitating their dissemination. Toxoplasma gondii tachyzoites divide using a unique form of cell division called endodyogeny. This process involves a single round of DNA replication, closed nuclear mitosis, and assembly of two daughter cells within a mother. In higher Eukaryotes, the four-subunit chromosomal passenger complex (CPC) (Aurora kinase B (ARKB)/INCENP/Borealin/Survivin) promotes chromosome bi-orientation by detaching incorrect kinetochore–microtubule attachments, playing an essential role in controlling cell division fidelity. Herein, we report the characterization of the Toxoplasma CPC (Aurora kinase 1 (Ark1)/INCENP1/INCENP2). We show that the CPC exhibits dynamic localization in a cell cycle-dependent manner. TgArk1 interacts with both TgINCENPs, with TgINCENP2 being essential for its translocation to the nucleus. While TgINCENP1 appears to be dispensable, interfering with TgArk1 or TgINCENP2 results in pronounced division and growth defects. Significant anti-cancer drug development efforts have focused on targeting human ARKB. Parasite treatment with low doses of hesperadin, a known inhibitor of human ARKB at higher concentrations, phenocopies the TgArk1 and TgINCENP2 mutants. Overall, our study provides new insights into the mechanisms underpinning cell cycle control in Apicomplexa, and highlights TgArk1 as potential drug target.     

Isolating <Emphasis Type="Italic">Escherichia coli</Emphasis> strains for recombinant protein production

Escherichia coli has been widely used for the production of recombinant proteins. To improve protein production yields in E. coli, directed engineering approaches have been commonly used. However, there are only few reported examples of the isolation of E. coli protein production strains using evolutionary approaches. Here, we first give an introduction to bacterial evolution and mutagenesis to set the stage for discussing how so far selection- and screening-based approaches have been used to isolate E. coli protein production strains. Finally, we discuss how evolutionary approaches may be used in the future to isolate E. coli strains with improved protein production characteristics.     

Membrane-shed vesicles from the parasite <Emphasis Type="Italic">Trichomonas vaginalis</Emphasis>: characterization and their association with cell interaction

Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogenital tract, where it remains extracellular and adheres to epithelial cells. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Despite the serious consequences associated with trichomoniasis disease, little is known about parasite or host factors involved in attachment of the parasite-to-host epithelial cells. Here, we report the identification of microvesicle-like structures (MVs) released by T. vaginalis. MVs are considered universal transport vehicles for intercellular communication as they can incorporate peptides, proteins, lipids, miRNA, and mRNA, all of which can be transferred to target cells through receptor–ligand interactions, fusion with the cell membrane, and delivery of a functional cargo to the cytoplasm of the target cell. In the present study, we demonstrated that T. vaginalis release MVs from the plasma and the flagellar membranes of the parasite. We performed proteomic profiling of these structures demonstrating that they possess physical characteristics similar to mammalian extracellular vesicles and might be selectively charged with specific protein content. In addition, we demonstrated that viable T. vaginalis parasites release large vesicles (LVs), membrane structures larger than 1 µm that are able to interact with other parasites and with the host cell. Finally, we show that both populations of vesicles present on the surface of T vaginalis are induced in the presence of host cells, consistent with a role in modulating cell interactions.     

<Emphasis Type="Italic">O</Emphasis>-GlcNAc transferase associates with the MCM2–7 complex and its silencing destabilizes MCM–MCM interactions

O-GlcNAcylation of proteins is governed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). The homeostasis of O-GlcNAc cycling is regulated during cell cycle progression and is essential for proper cellular division. We previously reported the O-GlcNAcylation of the minichromosome maintenance proteins MCM2, MCM3, MCM6 and MCM7. These proteins belong to the MCM2–7 complex which is crucial for the initiation of DNA replication through its DNA helicase activity. Here we show that the six subunits of MCM2–7 are O-GlcNAcylated and that O-GlcNAcylation of MCM proteins mainly occurs in the chromatin-bound fraction of synchronized human cells. Moreover, we identify stable interaction between OGT and several MCM subunits. We also show that down-regulation of OGT decreases the chromatin binding of MCM2, MCM6 and MCM7 without affecting their steady-state level. Finally, OGT silencing or OGA inhibition destabilizes MCM2/6 and MCM4/7 interactions in the chromatin-enriched fraction. In conclusion, OGT is a new partner of the MCM2–7 complex and O-GlcNAcylation homeostasis might regulate MCM2–7 complex by regulating the chromatin loading of MCM6 and MCM7 and stabilizing MCM/MCM interactions.     

Distinct functions of Crumbs regulating slit diaphragms and endocytosis in <Emphasis Type="Italic">Drosophila</Emphasis> nephrocytes

Mammalian podocytes, the key determinants of the kidney’s filtration barrier, differentiate from columnar epithelial cells and several key determinants of apical–basal polarity in the conventional epithelia have been shown to regulate podocyte morphogenesis and function. However, little is known about the role of Crumbs, a conserved polarity regulator in many epithelia, for slit-diaphragm formation and podocyte function. In this study, we used Drosophila nephrocytes as model system for mammalian podocytes and identified a conserved function of Crumbs proteins for cellular morphogenesis, nephrocyte diaphragm assembly/maintenance, and endocytosis. Nephrocyte-specific knock-down of Crumbs results in disturbed nephrocyte diaphragm assembly/maintenance and decreased endocytosis, which can be rescued by Drosophila Crumbs as well as human Crumbs2 and Crumbs3, which were both expressed in human podocytes. In contrast to the extracellular domain, which facilitates nephrocyte diaphragm assembly/maintenance, the intracellular FERM-interaction motif of Crumbs is essential for regulating endocytosis. Moreover, Moesin, which binds to the FERM-binding domain of Crumbs, is essential for efficient endocytosis. Thus, we describe here a new mechanism of nephrocyte development and function, which is likely to be conserved in mammalian podocytes.     

The ATP-binding cassette family: a structural perspective

The ATP-binding cassette family is one of the largest groupings of membrane proteins, moving allocrites across lipid membranes, using energy from ATP. In bacteria, they reside in the inner membrane and are involved in both uptake and export. In eukaryotes, these transporters reside in the cell’s internal membranes as well as in the plasma membrane and are unidirectional—out of the cytoplasm. The range of substances that these proteins can transport is huge, which makes them interesting for structure–function studies. Moreover, their abundance in nature has made them targets for structural proteomics consortia. There are eight independent structures for ATP-binding cassette transporters, making this one of the best characterised membrane protein families. Our understanding of the mechanism of transport across membranes and membrane protein structure in general has been enhanced by recent developments for this family.     

Cell-specific and lamin-dependent targeting of novel transmembrane proteins in the nuclear envelope

Nuclear envelope complexity is expanding with respect to identification of protein components. Here we test the validity of proteomics results that identified 67 novel predicted nuclear envelope transmembrane proteins (NETs) from liver by directly comparing 30 as tagged fusions using targeting assays. This confirmed 21 as NETs, but 4 only targeted in certain cell types, underscoring the complexity of interactions that tether NETs to the nuclear envelope. Four NETs accumulated at the nuclear rim in normal fibroblasts but not in fibroblasts lacking lamin A, suggesting involvement of lamin A in tethering them in the nucleus. However, intriguingly, for the NETs tested alternative mechanisms for nuclear envelope retention could be found in Jurkat cells that normally lack lamin A. This study expands by a factor of three the number of liver NETs analyzed, bringing the total confirmed to 31, and shows that several have multiple mechanisms for nuclear envelope retention.     

<Emphasis Type="Italic">O</Emphasis>-Mannosylation and human disease

Glycosylation of proteins is arguably the most prevalent co- and post-translational modification. It is responsible for increased heterogeneity and functional diversity of proteins. Here we discuss the importance of one type of glycosylation, specifically O-mannosylation and its relationship to a number of human diseases. The most widely studied O-mannose modified protein is alpha-dystroglycan (α-DG). Recent studies have focused intensely on α-DG due to the severity of diseases associated with its improper glycosylation. O-mannosylation of α-DG is involved in cancer metastasis, arenavirus entry, and multiple forms of congenital muscular dystrophy [1, 2]. In this review, we discuss the structural and functional characteristics of O-mannose-initiated glycan structures on α-DG, enzymes involved in the O-mannosylation pathway, and the diseases that are a direct result of disruptions within this pathway.