Arabidopsis membrane-anchored ubiquitin-fold (MUB) proteins localize a specific subset of ubiquitin-conjugating (E2) enzymes to the plasma membrane

The covalent attachment of ubiquitin (Ub) to various intracellular proteins plays important roles in altering the function, localization, processing, and degradation of the modified target. A minimal ubiquitylation pathway uses a three-enzyme cascade (E1, E2, and E3) to activate Ub and select target proteins for modification. Although diverse E3 families provide much of the target specificity, several factors have emerged recently that coordinate the subcellular localization of the ubiquitylation machinery. Here, we show that the family of membrane-anchored ubiquitin-fold (MUB) proteins recruits and docks specific E2s to the plasma membrane. Protein interaction screens with Arabidopsis MUBs revealed that interacting E2s are limited to a well defined subgroup that is phylogenetically related to human UbcH5 and yeast Ubc4/5 families. MUBs appear to interact noncovalently with an E2 surface opposite the active site that forms a covalent linkage with Ub. Bimolecular fluorescence complementation demonstrated that MUBs bind simultaneously to the plasma membrane via a prenyl tail and to the E2 in planta. These findings suggest that MUBs contribute subcellular specificity to ubiquitylation by docking the conjugation machinery to the plasma membrane.     

Solution structure of a membrane-anchored ubiquitin-fold (MUB) protein from Homo sapiens

The protein Bc059385, whose solution structure is reported here, is the human representative of a recently identified family of membrane-anchored ubiquitin-fold (MUB) proteins. Analysis of their similarity to ubiquitin indicates that homologous amino acid residues in MUBs form a hydrophobic surface very similar to the recognition patch surrounding Ile-44 in ubiquitin. This suggests that MUBs may interact with proteins containing an alpha-helical motif similar to those of some ubiquitin binding domains. A disordered loop common to MUBs may also provide a second protein interaction site. From the available data, it is probable that this protein is prenylated and associated with the membrane. With <20% identity to ubiquitin, the MUB family further expands the sequence space that maps to the beta-grasp fold, and adds membrane localization to its list of functional roles.     

Cell-cycle-dependent, differential prenylation of proteins

Isoprenylated proteins related to cell growth have been detected during proliferation. Since cholesterogenesis (isoprenoid synthesis) is mandatory for cell proliferation, the observation of a temporally coordinated protein prenylation during the cell division cycle might constitute obligatory processes in the signalling pathway for initiating DNA replication and/or in maintaining the growing state. We have found such a definitive cell-cycle-phase-dependent pattern of prenylation for various classes of cytosolic and nuclear matrix proteins in synchronized HepG2 cells. Characteristic [3H]mevalonate incorporation began to increase during mid-to-late G1, just after cholesterol synthesis reached its apex, and peaked just prior to or coincident with mid S. Incorporation then declined subsequent to S (during G2) as cells approached mitosis. Prior to the rise in mevalonate incorporation into proteins, during early-to-mid G1, steady-state [14C]acetate incorporation into chromatographically resolved cholesterogenic lipid intermediates displayed a maximum only into cholesterol. However, during the late-G1/S interval, a singular peak of 14C incorporation was found for the farnesyl moiety (farnesol/nerolidol plus farnesyl diphosphate). Except for the farnesyl moiety, none of the other polyisoprenoids detected by our procedures showed any fluctuation in 14C incorporation subsequent to mid G1. These results support the proposal that subsequent to peak cholesterol synthesis in early-to-mid G1, the generation of a cholesterol-pathway-dependent set of post-translationally modified, polyisoprenylated proteins could constitute an obligatory step leading to the duplication of the cellular genome, thereby impelling transit through the cell cycle. The well known high flux through cholesterogenesis in tumors, which manifests an intrinsic lack of sensitivity to feedback inhibition and operates continuously, is consonant with this proposal.     

The DAZL family proteins are PABP-binding proteins that regulate translation in germ cells

DAZL proteins are germ-cell-specific RNA-binding proteins essential for gametogenesis. The precise molecular role of these proteins in germ-cell development remains enigmatic; however, they appear to function in the cytoplasm. In order to directly address the function of vertebrate DAZL proteins, we have used Xenopus laevis oocytes as a model system. Here we demonstrate that members of this family, including Xdazl, mouse Dazl, human DAZL, human DAZ and human BOULE, have the ability to stimulate translation and function at the level of translation initiation. We show that DAZL proteins interact with poly(A)-binding proteins (PABPs), which are critical for the initiation of translation. Mapping and tethered function experiments suggest that these interactions are physiologically important. This leads to an attractive hypothesis whereby DAZL proteins activate translationally silent mRNAs during germ cell development through the direct recruitment of PABPs.     

Human cytoplasmic actin proteins are encoded by a multigene family

We characterized nine human actin genes that we isolated (Engel et al., Proc. Natl. Acad. Sci. U.S.A. 78:4674-4678, 1981) from a library of cloned human DNA. Measurements of the thermal stability of hybrids formed between each cloned actin gene and alpha-, beta-, and gamma-actin mRNA demonstrated that only one of the clones is most homologous to sarcomeric actin mRNA, whereas the remaining eight clones are most homologous to cytoplasmic actin mRNA. By the following criteria we show that these nine clones represent nine different actin gene loci rather than different alleles or different parts of a single gene: (i) the restriction enzyme maps of the coding regions are dissimilar; (ii) each clone contains sufficient coding region to encode all or most of an entire actin gene; and (iii) each clone contains sequences homologous to both the 5' and 3' ends of the coding region of a cloned chicken beta-actin cDNA. We conclude, therefore, that the human cytoplasmic actin proteins are encoded by a multigene family.     

Chemical site-selective prenylation of proteins

A direct thionation procedure allows conversion of allylic alcohols into the corresponding thiols, the products of which are immediately compatible with one-pot site-selective selenenyl sulfide mediated protein conjugation.     

The prenylation of proteins.

The prenylated proteins represent a newly discovered class of post-translationally modified proteins. The known prenylated proteins include the oncogene product p21ras and other low molecular weight GTP-binding proteins, the nuclear lamins, and the gamma subunit of the heterotrimeric G proteins. The modification involves the covalent attachment of a 15-carbon (farnesyl) or 20-carbon (geranylgeranyl) isoprenoid moiety in a thioether linkage to carboxyl terminal cysteine. The nature of the attached substituent is dependent on specific sequence information in the carboxyl terminus of the protein. In addition, prenylation entrains other posttranslational modifications forming a reaction pathway. In this article, we review our current understanding of the biochemical reactions involved in prenylation and discuss the possible role of this modification in the control of cellular functions such as protein maturation and cell growth.     

The cataract-associated protein TMEM114, and TMEM235, are glycosylated transmembrane proteins that are distinct from claudin family members

A novel gene, TMEM114, was annotated as a member of the claudin gene family and was subsequently associated as a cause of autosomal dominant cataract because of a translocation in its putative promoter. Our bioinformatic and molecular analyses of TMEM114, and the closely related TMEM235, demonstrate that these proteins are more closely related to members of the voltage dependent calcium channel gamma subunit family. TMEM114 and TMEM235 differed from claudins in terms of localisation in polarised epithelial cells and by the presence of N-linked glycans. By gene expression knockdown in Xenopus tropicalis we also demonstrate a role for Tmem114 in eye development.     

ORMDL proteins are a conserved new family of endoplasmic reticulum membrane proteins

Background  

Annotations of completely sequenced genomes reveal that nearly half of the genes identified are of unknown function, and that some belong to uncharacterized gene families. To help resolve such issues, information can be obtained from the comparative analysis of homologous genes in model organisms.     

Ultrasensitivity in multisite phosphorylation of membrane-anchored proteins

Cellular signaling is initially confined to the plasma membrane, where the cytoplasmic tails of surface receptors and other membrane-anchored proteins are phosphorylated in response to ligand binding. These proteins often contain multiple phosphorylation sites that are regulated by membrane-confined enzymes. Phosphorylation of these proteins is thought to be tightly regulated, because they initiate and regulate signaling cascades leading to cellular activation, yet how their phosphorylation is regulated is poorly understood. Ultrasensitive or switchlike responses in their phosphorylation state are not expected because the modifying enzymes are in excess. Here, we describe a novel mechanism of ultrasensitivity exhibited by multisite membrane-anchored proteins, but not cytosolic proteins, even when enzymes are in excess. The mechanism underlying this concentration-independent ultrasensitivity is the local saturation of a single enzyme by multiple sites on the substrate. Local saturation is a passive process arising from slow membrane diffusion, steric hindrances, and multiple sites, and therefore may be widely applicable. Critical to this ultrasensitivity is the brief enzymatic inactivation that follows substrate modification. Computations are presented using ordinary differential equations and stochastic spatial simulations. We propose a new role, to our knowledge, for multisite membrane-anchored proteins, discuss experiments that can be used to probe the model, and relate our findings to previous theoretical work.     

The synucleins are a family of redox-active copper binding proteins

Thermodynamic studies in conjunction with EPR confirm that α-synuclein, β-synuclein, and γ-synuclein bind copper(II) in a high affinity 1:1 stoichiometry. γ-Synuclein demonstrates the highest affinity, in the picomolar range, while α-synuclein and β-synuclein both bind copper(II) with nanomolar affinity. The copper center on all three proteins demonstrates reversible or partly reversible redox cycling. Various mutations show that the primary coordinating ligand for copper(II) is located within the N-terminal regions between residues 2-9. There is also a contribution from the C-terminus in conjunction with the histidine at position 50 in α-synuclein and position 65 in β-synuclein, although these regions appear to have little effect on overall coordination stability. These histidines and the C-terminus, however, appear to be critical to the redox engine of the proteins.     

Alveolins, a new family of cortical proteins that define the protist infrakingdom Alveolata

Alveolates are a recently recognized group of unicellular eukaryotes that unites disparate protists including apicomplexan parasites (which cause malaria and toxoplasmosis), dinoflagellate algae (which cause red tides and are symbionts in many corals), and ciliates (which are microscopic predators and common rumen symbionts). Gene sequence trees provide robust support for the alveolate alliance, but beyond the common presence of membranous sacs (alveoli) subtending the plasma membrane, the group has no unifying morphological feature. We describe a family of proteins, alveolins, associated with these membranous sacs in apicomplexa, dinoflagellates, and ciliates. Alveolins contain numerous simple peptide repeats and are encoded by multigene families. We generated antibodies against a peptide motif common to all alveolins and identified a range of apparently abundant proteins in apicomplexa, dinoflagellates, and ciliates. Immunolocalization reveals that alveolins are associated exclusively with the cortical regions of apicomplexa, dinoflagellates, and ciliates where the alveolar sacs occur. Alveolins are the first molecular nexus between the unifying structures that defines this eukaryotic group. They provide an excellent opportunity to explore the exceptional compartment that was apparently the key to a remarkable diversification of unique protists that occupy a wide array of lifestyle niches.     

The Saccharomyces cerevisiae YLL012/YEH1, YLR020/YEH2, and TGL1 genes encode a novel family of membrane-anchored lipases that are required for steryl ester hydrolysis

Sterol homeostasis in eukaryotic cells relies on the reciprocal interconversion of free sterols and steryl esters. The formation of steryl esters is well characterized, but the mechanisms that control steryl ester mobilization upon cellular demand are less well understood. We have identified a family of three lipases of Saccharomyces cerevisiae that are required for efficient steryl ester mobilization. These lipases, encoded by YLL012/YEH1, YLR020/YEH2, and TGL1, are paralogues of the mammalian acid lipase family, which is composed of the lysosomal acid lipase, the gastric lipase, and four novel as yet uncharacterized human open reading frames. Lipase triple-mutant yeast cells are completely blocked in steryl ester hydrolysis but do not affect the mobilization of triacylglycerols, indicating that the three lipases are required for steryl ester mobilization in vivo. Lipase single mutants mobilize steryl esters to various degrees, indicating partial functional redundancy of the three gene products. Lipase double-mutant cells in which the third lipase is expressed from the inducible GAL1 promoter have greatly reduced steady-state levels of steryl esters, indicating that overexpression of any of the three lipases is sufficient for steryl ester mobilization in vivo. The three yeast enzymes constitute a novel class of membrane-anchored lipases that differ in topology and subcellular localization.     

Signaling pathways are focused at specialized regions of the plasma membrane by scaffolding proteins of the MAGUK family.

The MAGUKs (membrane-associated guanylate kinase homologs) are a family of proteins that act as molecular scaffolds for signaling pathway components at the plasma membrane of animal cells. They are localized in and required for the formation of several types of cell junctions, including epithelial tight and septate junctions as well as synaptic and neuromuscular junctions. They are also localized at the plasma membrane of other cell types, including erythrocytes, where they contribute to cell shape maintenance. MAGUKs function mainly by binding directly to the cytoplasmic termini of transmembrane proteins as well as to other signal transduction proteins. They appear to hold together elements of individual signaling pathways, thereby contributing to the efficiency and specificity of signaling interactions while simultaneously maintaining the structural specializations of the plasma membrane. BioEssays 1999;21:912-921.     

PAT proteins,an ancient family of lipid droplet proteins that regulate cellular lipid stores

The PAT family of lipid droplet proteins includes 5 members in mammals: perilipin, adipose differentiation-related protein (ADRP), tail-interacting protein of 47 kDa (TIP47), S3–12, and OXPAT. Members of this family are also present in evolutionarily distant organisms, including insects, slime molds and fungi. All PAT proteins share sequence similarity and the ability to bind intracellular lipid droplets, either constitutively or in response to metabolic stimuli, such as increased lipid flux into or out of lipid droplets. Positioned at the lipid droplet surface, PAT proteins manage access of other proteins (lipases) to the lipid esters within the lipid droplet core and can interact with cellular machinery important for lipid droplet biogenesis. Genetic variations in the gene for the best-characterized of the mammalian PAT proteins, perilipin, have been associated with metabolic phenotypes, including type 2 diabetes mellitus and obesity. In this review, we discuss how the PAT proteins regulate cellular lipid metabolism both in mammals and in model organisms.