Transmembrane proteins are not required for early stages of nuclear envelope assembly

All identified membrane fusion proteins are transmembrane proteins. In the present study, we explored the post-mitotic reassembly of the NE (nuclear envelope). The proteins that drive membrane rearrangements in NE assembly remain unknown. To determine whether transmembrane proteins are prerequisite components of this fusion machinery, we have focused on nuclear reconstitution in a cell-free system. Mixing of soluble interphase cytosolic extract and MV (membrane vesicles) from amphibian eggs with chromatin results in the formation of functional nuclei. We replaced MV and cytosol with protein-free phosphatidylcholine LS (liposomes) that were pre-incubated with interphase cytosol. While later stages of NE assembly yielding functional nucleus did not proceed without integral proteins of MV, LS-associated cytosolic proteins were sufficient to reconstitute membrane targeting to the chromatin and GTP-dependent lipid mixing. Binding involved LS-associated A-type lamin, and fusion involved Ran GTPase. Thus in contrast with post-fusion stages, fusion initiation in NE assembly, like membrane remodelling in budding and fission, does not require transmembrane proteins.     

The conserved transmembrane nucleoporin NDC1 is required for nuclear pore complex assembly in vertebrate cells

Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in the nuclear envelope (NE), through which exchange of molecules between the nucleus and cytosol occurs. Biogenesis of NPCs is complex and poorly understood. In particular, almost nothing is known about how NPCs are anchored in the NE. Here, we characterize vertebrate NDC1--a transmembrane nucleoporin conserved between yeast and metazoans. We show by RNA interference (RNAi) and biochemical depletion that NDC1 plays an important role in NPC and NE assembly in vivo and in vitro. RNAi experiments suggest a functional link between NDC1 and the soluble nucleoporins Nup93, Nup53, and Nup205. Importantly, NDC1 interacts with Nup53 in vitro. This suggests that NDC1 function involves forming a link between the NE membrane and soluble nucleoporins, thereby anchoring the NPC in the membrane.     

Respiratory chain complex I is essential for sexual development in neurospora and binding of iron sulfur clusters are required for enzyme assembly

We have cloned and disrupted in vivo, by repeat-induced point mutations, the nuclear gene coding for an iron sulfur subunit of complex I from Neurospora crassa, homologue of the mammalian TYKY protein. Analysis of the obtained mutant nuo21.3c revealed that complex I fails to assemble. The peripheral arm of the enzyme is disrupted while its membrane arm accumulates. Furthermore, mutated 21.3c-kD proteins, in which selected cysteine residues were substituted with alanines or serines, were expressed in mutant nuo21. 3c. The phenotypes of these strains regarding the formation of complex I are similar to that of the original mutant, indicating that binding of iron sulfur centers to protein subunits is a prerequisite for complex I assembly. Homozygous crosses of nuo21.3c strain, and of other complex I mutants, are unable to complete sexual development. The crosses are blocked at an early developmental stage, before fusion of the nuclei of opposite mating types. This phenotype can be rescued only by transformation with the intact gene. Our results suggest that this might be due to the compromised capacity of complex I-defective strains in energy production.     

Novel conserved motifs in Rev1 C-terminus are required for mutagenic DNA damage tolerance

The genes encoding Rev1 and DNA polymerase zeta (Rev3/Rev7) are together required for the vast majority of DNA damage-induced mutations in eukaryotes from yeast to humans. Here, we provide insight into the critical role that the Saccharomyces cerevisiae Rev1 C-terminus plays in the process of mutagenic DNA damage tolerance. The Rev1 C-terminus was previously thought to be poorly conserved and therefore not likely to be important for mediating protein-protein interactions. However, through comprehensive alignments of the Rev1 C-terminus, we have identified novel and hitherto unrecognized conserved motifs that we show play an essential role in REV1-dependent survival and mutagenesis in S. cerevisiae, likely in its post-replicative gap-filling mode. We further show that the minimal C-terminal fragment of Rev1 containing these highly conserved motifs is sufficient to interact with Rev7.     

Residues within a conserved amino acid motif of domains 1 and 4 of VCAM- 1 are required for binding to VLA-4

Vascular cell adhesion molecule 1 (VCAM-1), a member of the Ig superfamily originally identified on activated endothelium, binds to the integrin very late antigen-4 (VLA-4), also known as alpha 4 beta 1 or CD49d/CD29, to support cell-cell adhesion. Studies based on cell adhesion to two alternatively spliced forms of VCAM-1 or to chimeric molecules generated from them and intercellular adhesion molecule-1 (ICAM-1) have demonstrated two VLA-4 binding sites on the predominate form of VCAM-1. Here, we studied VLA-4-dependent adhesion of the lymphoid tumor cell line Ramos to cells expressing wild type and mutant forms of VCAM-1. Results based on domain deletion mutants demonstrated the existence and independence of two VLA-4-binding sites located in the first and fourth domains of VCAM-1. Results based on amino acid substitution mutants demonstrated that residues within a linear sequence of six amino acids found in both domain 1 and 4 were required for VLA-4 binding to either domain. Five of these amino acids represent a conserved motif also found in ICAM domains. We propose that integrin binding to these Ig-like domains depends on residues within this conserved motif. Specificity of integrin binding to Ig-like domains may be regulated by a set of nonconserved residues distinct from the conserved motif.     

Glycosphingolipids are required for sorting melanosomal proteins in the Golgi complex.

Although glycosphingolipids are ubiquitously expressed and essential for multicellular organisms, surprisingly little is known about their intracellular functions. To explore the role of glycosphingolipids in membrane transport, we used the glycosphingolipid-deficient GM95 mouse melanoma cell line. We found that GM95 cells do not make melanin pigment because tyrosinase, the first and rate-limiting enzyme in melanin synthesis, was not targeted to melanosomes but accumulated in the Golgi complex. However, tyrosinase-related protein 1 still reached melanosomal structures via the plasma membrane instead of the direct pathway from the Golgi. Delivery of lysosomal enzymes from the Golgi complex to endosomes was normal, suggesting that this pathway is not affected by the absence of glycosphingolipids. Loss of pigmentation was due to tyrosinase mislocalization, since transfection of tyrosinase with an extended transmembrane domain, which bypassed the transport block, restored pigmentation. Transfection of ceramide glucosyltransferase or addition of glucosylsphingosine restored tyrosinase transport and pigmentation. We conclude that protein transport from Golgi to melanosomes via the direct pathway requires glycosphingolipids.     

Immunogold quantification of amino acids and proteins in complex subcellular compartments

An increasing number of imaging techniques are in use to study the localization of molecules involved in cell-to-cell signaling. Here we describe the use of immunogold procedures to detect and quantify molecules on electron micrographs. To measure the areas of the subcellular compartments under investigation, the protocol uses an overlay screen with an array of regularly spaced points. On the basis of this, the densities of the gold-labeled molecules can be calculated. Despite the limited lateral resolution of the immunogold method as used by many investigators ( approximately 30 nm), it is possible to measure the content of molecules associated with tiny tissue compartments, e.g., synaptic vesicles and different types of membrane, such as plasma membranes and vesicle membranes. The quantification protocol can be carried out without using computer programs. The entire protocol can be completed in approximately 15 d.     

A conserved signal and GTPase complex are required for the ciliary transport of polycystin-1

Primary cilia regulate epithelial differentiation and organ function. Failure of mutant polycystins to localize to cilia abolishes flow-stimulated calcium signaling and causes autosomal dominant polycystic kidney disease. We identify a conserved amino acid sequence, KVHPSST, in the C-terminus of polycystin-1 (PC1) that serves as a ciliary-targeting signal. PC1 binds a multimeric protein complex consisting of several GTPases (Arf4, Rab6, Rab11) and the GTPase-activating protein (GAP), ArfGAP with SH3 domain, ankyrin repeat and PH domain 1 (ASAP1) in the Golgi, which facilitates vesicle budding and Golgi exocytosis. A related N-terminal ciliary-targeting sequence in polycystin-2 similarly binds Arf4. Deletion of the extreme C-terminus of PC1 ablates Arf4 and ASAP1 binding and prevents ciliary localization of an integral membrane CD16.7-PC1 chimera. Interactions are confirmed for chimeric and endogenous proteins through quantitated in vitro and cell-based approaches. PC1 also complexes with Rab8; knockdown of trafficking regulators Arf4 or Rab8 functionally blocks CD16.7-PC1 trafficking to cilia. Mutations in rhodopsin disrupt a similar signal and cause retinitis pigmentosa, while Bardet-Biedl syndrome, primary open-angle glaucoma, and tumor cell invasiveness are linked to dysregulation of ASAP1 or Rab8 or its effectors. In this paper, we provide evidence for a conserved GTPase-dependent ciliary-trafficking mechanism that is shared between epithelia and neurons, and is essential in ciliary-trafficking and cell homeostasis.     

N-terminal basic amino acids are not required for translocation and processing of preproparathyroid hormone

An N-terminal deletion mutant of preproparathyroid hormone that contains a single basic amino acid, lysine, in the N-terminal domain of the signal peptide is translocated across the endoplasmic reticulum membrane similarly to intact preproparathyroid hormone. To examine the function of charged residues preceeding the hydrophobic core, the lysine was replaced by an uncharged (methionine) or negatively charged (glutamic acid) amino acid. The translocational activity of the mutant signal peptides was assayed in a reticulocyte lysate system containing chicken oviduct microsomal membranes. Altering the net charge of the N-terminal domain did not abolish signal sequence activity, although the efficiency of translocation was decreased for the mutant with a glutamic acid substitution. Posttranslational, ribosome independent, translocation was observed for all the mutants tested, with the same dependence on N-terminal charge but with much lower efficiency than cotranslational translocation. These studies show that the presence of basic amino acids in the N-terminal domain of a eukaryotic signal sequence is not required for its activity.     

Interaction of DNA with clusters of amino acids in proteins

Protein-DNA interactions facilitate the fundamental functions of living cells and are universal in all living organisms. Several investigations have been carried out, essentially identifying pairs of interactions between the amino acid residues in proteins and the bases in DNA. In the present study, we have detected the recognition motifs that may constitute a cluster of spatially interacting residues in proteins, which interact with the bases of DNA. Graph spectral algorithm has been used to detect side chain clusters comprising Arg, Lys, Asn, Gln and aromatic residues from proteins interacting with DNA. We find that the interaction of proteins with DNA is through clusters in about half of the proteins in the dataset and through individual residues in the rest. Furthermore, inspection of the clusters has revealed additional interactions in a few cases, which have not been reported earlier. The geometry of the interaction between the DNA base and the protein residue is quantified by the distance d and the angle theta. These parameters have been identified for the cation-pi/H-bond stair motif that was reported earlier. Among the Arg, Lys, Asn and Gln residues, the range of (d, theta) values of the interacting Arg clearly falls into the cation-pi and the hydrogen bond interactions of the 'cation-pi/H-bond' stair motif. Analysis of the cluster composition reveals that the Arg residue is predominant than the Lys, Asn and Gln residues. The clusters are classified into Type I and Type II based on the presence or absence of aromatic residues (Phe, Tyr) in them. Residue conservation in these clusters has been examined. Apart from the conserved residues identified previously, a few more residues mainly Phe, Tyr and Arg have also been identified as conserved and interactive with the DNA. Interestingly, a few residues that are parts of interacting clusters and do not interact directly with the DNA have also been conserved. This emphasizes the importance of recognizing the protein side chain cluster motifs interacting with the DNA, which could serve as signatures of protein-DNA recognition in the families of DNA binding proteins.     

The conserved proteins CHE-12 and DYF-11 are required for sensory cilium function in Caenorhabditis elegans

Sensory neuron cilia are evolutionarily conserved dendritic appendages that convert environmental stimuli into neuronal activity. Although several cilia components are known, the functions of many remain uncharacterized. Furthermore, the basis of morphological and functional differences between cilia remains largely unexplored. To understand the molecular basis of cilia morphogenesis and function, we studied the Caenorhabditis elegans mutants che-12 and dyf-11. These mutants fail to concentrate lipophilic dyes from their surroundings in sensory neurons and are chemotaxis defective. In che-12 mutants, sensory neuron cilia lack distal segments, while in dyf-11 animals, medial and distal segments are absent. CHE-12 and DYF-11 are conserved ciliary proteins that function cell-autonomously and are continuously required for maintenance of cilium morphology and function. CHE-12, composed primarily of HEAT repeats, may not be part of the intraflagellar transport (IFT) complex and is not required for the localization of some IFT components. DYF-11 undergoes IFT-like movement and may function at an early stage of IFT-B particle assembly. Intriguingly, while DYF-11 is expressed in all C. elegans ciliated neurons, CHE-12 expression is restricted to some amphid sensory neurons, suggesting a specific role in these neurons. Our results provide insight into general and neuron-specific aspects of cilium development and function.     

A CCAAT DNA binding factor consisting of two different components that are both required for DNA binding

A CCAAT-binding activity present in nuclear extracts of rat liver and NIH 3T3 fibroblasts was purified using, as assay, DNA binding to a segment of the mouse alpha 2(I) collagen promoter. The activity consists of two components, designated factors A and B, which are separated by ion exchange chromatography on either Mono Q or Mono S columns. Factor A is heat-sensitive, whereas factor B is heat-resistant. Both factors are required for DNA binding and both are present in the DNA protein complex. The A + B complex was extensively purified by heparin-agarose and sequence-specific affinity chromatography. The Mr of factor A is 39,000, whereas the Mr of factor B is 41,000 as determined by renaturation of a highly purified preparation after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Competition experiments indicate that this CCAAT-binding complex has a DNA sequence specificity that is different from those of other CCAAT-binding proteins.     

The WD40 repeats of FANCL are required for Fanconi anemia core complex assembly

Fanconi anemia (FA) is an autosomal recessive disorder characterized by aplastic anemia, cancer susceptibility, and cellular sensitivity to mitomycin C. Eight of the 11 cloned Fanconi anemia gene products (FANCA, -B, -C, -E, -F, -G, -L, and -M) form a multisubunit nuclear complex (FA core complex) required for monoubiquitination of a downstream FA protein, FANCD2. FANCL, which possesses three WD40 repeats and a plant homeodomain (PHD), is the putative E3 ubiquitin ligase subunit of the FA complex. Here, we demonstrate that the WD40 repeats of FANCL are required for interaction with other subunits of the FA complex. The PHD is dispensable for this interaction, although it is required for FANCD2 mono-ubiquitination. The PHD of FANCL also shares sequence similarity to the canonical RING finger of c-CBL, including a conserved tryptophan required for E2 binding by c-CBL. Mutation of this tryptophan in the FANCL PHD significantly impairs in vivo mono-ubiquitination of FANCD2 and in vitro auto-ubiquitination activity, and partially impairs restoration of mitomycin C resistance. We propose a model in which FANCL, via its WD40 region, binds the FA complex and, via its PHD, recruits an as-yet-unidentified E2 for mono-ubiquitination of FANCD2.