A family of small coiled-coil-forming proteins functioning at the late endosome in yeast

The multispanning membrane protein Ste6, a member of the ABC-transporter family, is transported to the yeast vacuole for degradation. To identify functions involved in the intracellular trafficking of polytopic membrane proteins, we looked for functions that block Ste6 transport to the vacuole upon overproduction. In our screen, we identified several known vacuolar protein sorting (VPS) genes (SNF7/VPS32, VPS4, and VPS35) and a previously uncharacterized open reading frame, which we named MOS10 (more of Ste6). Sequence analysis showed that Mos10 is a member of a small family of coiled-coil-forming proteins, which includes Snf7 and Vps20. Deletion mutants of all three genes stabilize Ste6 and show a "class E vps phenotype." Maturation of the vacuolar hydrolase carboxypeptidase Y was affected in the mutants and the endocytic tracer FM4-64 and Ste6 accumulated in a dot or ring-like structure next to the vacuole. Differential centrifugation experiments demonstrated that about half of the hydrophilic proteins Mos10 and Vps20 was membrane associated. The intracellular distribution was further analyzed for Mos10. On sucrose gradients, membrane-associated Mos10 cofractionated with the endosomal t-SNARE Pep12, pointing to an endosomal localization of Mos10. The growth phenotypes of the mutants suggest that the "Snf7-family" members are involved in a cargo-specific event.     

Arabidopsis P-protein filament formation requires both AtSEOR1 and AtSEOR2

The structure-function relationship of proteinaceous filaments in sieve elements has long been a source of investigation in order to understand their role in the biology of the phloem. Two phloem filament proteins AtSEOR1 (At3g01680.1) and AtSEOR2 (At3g01670.1) in Arabidopsis have been identified that are required for filament formation. Immunolocalization experiments using a phloem filament-specific monoclonal antibody in the respective T-DNA insertion mutants provided an initial indication that both proteins are necessary to form phloem filaments. To investigate the relationship between these two proteins further, green fluorescent protein (GFP)-AtSEO fusion proteins were expressed in Columbia wild-type and T-DNA insertion mutants. Analysis of these mutants by confocal microscopy confirmed that phloem filaments could only be detected in the presence of both proteins, indicating that despite significant sequence homology the proteins are not functionally redundant. Individual phloem filament protein subunits of AtSEOR1 and AtSEOR2 were capable of forming homodimers, but not heterodimers in a yeast two-hybrid system. The absence of phloem filaments in phloem sieve elements did not result in gross alterations of plant phenotype or affect basal resistance to green peach aphid (Myzus persicae).     

Degradation of Mos by the N-terminal proline (Pro2)-dependent ubiquitin pathway on fertilization of Xenopus eggs: possible significance of natural selection for Pro2 in Mos.

The c-mos proto-oncogene product (Mos), an essential component of the cytostatic factor responsible for meiotic arrest in vertebrate eggs, undergoes specific proteolysis soon after fertilization or activation of Xenopus eggs. To determine the degradation pathway of Mos on egg activation, various Mos mutants were expressed in Xenopus eggs and their degradation on egg activation was examined. Mos degradation absolutely required its penultimate proline (Pro2) residue and dephosphorylation of the adjacent serine (Ser3) residue. These degradation signals were essentially the same as those of Mos in meiosis I of Xenopus oocyte maturation, where Mos has been shown to be degraded by the 'second-codon rule'-based ubiquitin pathway. To test whether Mos degradation on egg activation is also mediated by the ubiquitin pathway, we attempted to identify and abrogate a specific ubiquitination site(s) in Mos. We show that the major ubiquitination site in Mos is a Lys34 residue and that replacement of this residue with a non-ubiquitinatable Arg residue markedly enhances the stability of Mos on egg activation. These results indicate that the degradation of Mos on egg activation or fertilization is mediated primarily by the N-terminal Pro2-dependent ubiquitin pathway, as in meiosis I of oocyte maturation. The N-terminal Pro2 residue of Mos appears to be naturally selected primarily for its degradation on fertilization, rather than that in meiosis I.     

Deletions in epidermal keratins leading to alterations in filament organization in vivo and in intermediate filament assembly in vitro

To investigate the sequences important for assembly of keratins into 10- nm filaments, we used a combined approach of (a) transfection of mutant keratin cDNAs into epithelial cells in vivo, and (b) in vitro assembly of mutant and wild-type keratins. Keratin K14 mutants missing the nonhelical carboxy- and amino-terminal domains not only integrated without perturbation into endogenous keratin filament networks in vivo, but they also formed 10-nm filaments with K5 in vitro. Surprisingly, keratin mutants missing the highly conserved L L E G E sequence, common to all intermediate filament proteins and found at the carboxy end of the alpha-helical rod domain, also assembled into filaments with only a somewhat reduced efficiency. Even a carboxy K14 mutant missing approximately 10% of the rod assembled into filaments, although in this case filaments aggregated significantly. Despite the ability of these mutants to form filaments in vitro, they often perturbed keratin filament organization in vivo. In contrast, small truncations in the amino-terminal end of the rod domain more severely disrupted the filament assembly process in vitro as well as in vivo, and in particular restricted elongation. For both carboxy and amino rod deletions, the more extensive the deletion, the more severe the phenotype. Surprisingly, while elongation could be almost quantitatively blocked with large mutations, tetramer formation and higher ordered lateral interactions still occurred. Collectively, our in vitro data (a) provide a molecular basis for the dominance of our mutants in vivo, (b) offer new insights as to why different mutants may generate different phenotypes in vivo, and (c) delineate the limit sequences necessary for K14 to both incorporate properly into a preexisting keratin filament network in vivo and assemble efficiently into 10-nm keratin filaments in vitro.     

A genomic screen for yeast vacuolar membrane ATPase mutants

V-ATPases acidify multiple organelles, and yeast mutants lacking V-ATPase activity exhibit a distinctive set of growth defects. To better understand the requirements for organelle acidification and the basis of these growth phenotypes, approximately 4700 yeast deletion mutants were screened for growth defects at pH 7.5 in 60 mm CaCl(2). In addition to 13 of 16 mutants lacking known V-ATPase subunits or assembly factors, 50 additional mutants were identified. Sixteen of these also grew poorly in nonfermentable carbon sources, like the known V-ATPase mutants, and were analyzed further. The cwh36Delta mutant exhibited the strongest phenotype; this mutation proved to disrupt a previously uncharacterized V-ATPase subunit. A small subset of the mutations implicated in vacuolar protein sorting, vps34Delta, vps15Delta, vps45Delta, and vps16Delta, caused both Vma- growth phenotypes and lower V-ATPase activity in isolated vacuoles, as did the shp1Delta mutation, implicated in both protein sorting and regulation of the Glc7p protein phosphatase. These proteins may regulate V-ATPase targeting and/or activity. Eight mutants showed a Vma- growth phenotype but no apparent defect in vacuolar acidification. Like V-ATPase-deficient mutants, most of these mutants rely on calcineurin for growth, particularly at high pH. A requirement for constitutive calcineurin activation may be the predominant physiological basis of the Vma- growth phenotype.     

Genetic dissection of novel myopathy models reveals a role of CapZα and Leiomodin 3 during myofibril elongation

Myofibrils within skeletal muscle are composed of sarcomeres that generate force by contraction when their myosin-rich thick filaments slide past actin-based thin filaments. Although mutations in components of the sarcomere are a major cause of human disease, the highly complex process of sarcomere assembly is not fully understood. Current models of thin filament assembly highlight a central role for filament capping proteins, which can be divided into three protein families, each ascribed with separate roles in thin filament assembly. CapZ proteins have been shown to bind the Z-disc protein α-actinin to form an anchoring complex for thin filaments and actin polymerisation. Subsequent thin filaments extension dynamics are thought to be facilitated by Leiomodins (Lmods) and thin filament assembly is concluded by Tropomodulins (Tmods) that specifically cap the pointed end of thin filaments. To study thin filament assembly in vivo, single and compound loss-of-function zebrafish mutants within distinct classes of capping proteins were analysed. The generated lmod3- and capza1b-deficient zebrafish exhibited aspects of the pathology caused by variations in their human orthologs. Although loss of the analysed main capping proteins of the skeletal muscle, capza1b, capza1a, lmod3 and tmod4, resulted in sarcomere defects, residual organised sarcomeres were formed within the assessed mutants, indicating that these proteins are not essential for the initial myofibril assembly. Furthermore, detected similarity and location of myofibril defects, apparent at the peripheral ends of myofibres of both Lmod3- and CapZα-deficient mutants, suggest a function in longitudinal myofibril growth for both proteins, which is molecularly distinct to the function of Tmod4.     

Genes for directing vacuolar morphogenesis in Saccharomyces cerevisiae. I. Isolation and characterization of two classes of vam mutants.

We identified nine VAM genes (for vacuolar morphology) by genetic analyses on mutants with defective vacuolar morphologies and assembly in the yeast Saccharomyces cerevisiae. The nine VAM genes were classified into two classes according to the mutant phenotypes. The class I vam mutants (vam1, vam5, vam8, and vam9) show a few small vesicles that are stained with histochemical markers for the vacuolar compartment. They also have defects in the maturation of vacuolar marker proteins, and their growth is hypersensitive to high concentrations of CaCl2 or a temperature of 37 degrees C. There are apparent genetic overlaps among the class I vam mutations and other mutations including cls, end, pep, and vps, which have been shown to be involved in the expression of the vacuolar functions. The class II vam mutants (vam2, vam3, vam4, vam6, and vam7) contain numerous small vesicles stained with the vacuolar histochemical markers and mature forms of the vacuolar proteins and do not show any apparent growth defects in the presence of CaCl2 or at 37 degrees C.     

ORGANIZATION AND BREAKDOWN OF THE PROTOPLAST DURING MATURATION OF PINE TRACHEIDS

The protoplast of maturing axial tracheids in the secondary xylem of shortleaf pine (Pinus echinata Mill.) was studied by transmission and scanning electron microscopy. The mature protoplast is differentiated into two interconnected components: (1) the commonly observed peripheral layer lining the secondary cell wall, and (2) an elaborate reticulum of cytoplasmic filaments and placoids within the central vacuole. The reticulum provides an extensive surface area of vacuolar membranes for rapid exchange of nutrients and metabolites with the vacuolar sap, which is envisaged to function as a vital medium during the period of secondary cell wall synthesis. The breakdown of the protoplast which terminates tracheid maturation is associated with poorly defined alterations of the vacuolar membranes. This is indicated by increased formation of cytoplasmic spherules and membraneous vesicles which may be portions of separated vacuolar membrane during early stages of degradation. Autolysis is supposed to occur when the cytoplasm is exposed to the vacuolar sap after rupture and separation of the vacuolar membranes. The Gomori acid phosphatase technique as combined with electron microscopy produced no evidence of autolysosomal segresomes in strands of intravacuolar reticulum of the cytoplasm.     

Actin filaments play a critical role in vacuolar trafficking at the Golgi complex in plant cells

Actin filaments are thought to play an important role in intracellular trafficking in various eukaryotic cells. However, their involvement in intracellular trafficking in plant cells has not been clearly demonstrated. Here, we investigated the roles actin filaments play in intracellular trafficking in plant cells using latrunculin B (Lat B), an inhibitor of actin filament assembly, or actin mutants that disrupt actin filaments when overexpressed. Lat B and actin2 mutant overexpression inhibited the trafficking of two vacuolar reporter proteins, sporamin:green fluorescent protein (GFP) and Arabidopsis thaliana aleurain-like protein:GFP, to the central vacuole; instead, a punctate staining pattern was observed. Colocalization experiments with various marker proteins indicated that these punctate stains corresponded to the Golgi complex. The A. thaliana vacuolar sorting receptor VSR-At, which mainly localizes to the prevacuolar compartment, also accumulated at the Golgi complex in the presence of Lat B. However, Lat B had no effect on the endoplasmic reticulum (ER) to Golgi trafficking of sialyltransferase or retrograde Golgi to ER trafficking. Lat B also failed to influence the Golgi to plasma membrane trafficking of H+-ATPase:GFP or the secretion of invertase:GFP. Based on these observations, we propose that actin filaments play a critical role in the trafficking of proteins from the Golgi complex to the central vacuole.     

Different domains of the essential GTPase Cdc42p required for growth and development of Saccharomyces cerevisiae

In budding yeast, the Rho-type GTPase Cdc42p is essential for cell division and regulates pseudohyphal development and invasive growth. Here, we isolated novel Cdc42p mutant proteins with single-amino-acid substitutions that are sufficient to uncouple functions of Cdc42p essential for cell division from regulatory functions required for pseudohyphal development and invasive growth. In haploid cells, Cdc42p is able to regulate invasive growth dependent on and independent of FLO11 gene expression. In diploid cells, Cdc42p regulates pseudohyphal development by controlling pseudohyphal cell (PH cell) morphogenesis and invasive growth. Several of the Cdc42p mutants isolated here block PH cell morphogenesis in response to nitrogen starvation without affecting morphology or polarity of yeast form cells in nutrient-rich conditions, indicating that these proteins are impaired for certain signaling functions. Interaction studies between development-specific Cdc42p mutants and known effector proteins indicate that in addition to the p21-activated (PAK)-like protein kinase Ste20p, the Cdc42p/Rac-interactive-binding domain containing Gic1p and Gic2p proteins and the PAK-like protein kinase Skm1p might be further effectors of Cdc42p that regulate pseudohyphal and invasive growth.     

Cell Envelope Components Influencing Filament Length in the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120

Heterocyst-forming cyanobacteria grow as chains of cells (known as trichomes or filaments) that can be hundreds of cells long. The filament consists of individual cells surrounded by a cytoplasmic membrane and peptidoglycan layers. The cells, however, share a continuous outer membrane, and septal proteins, such as SepJ, are important for cell-cell contact and filament formation. Here, we addressed a possible role of cell envelope components in filamentation, the process of producing and maintaining filaments, in the model cyanobacterium Anabaena sp. strain PCC 7120. We studied filament length and the response of the filaments to mechanical fragmentation in a number of strains with mutations in genes encoding cell envelope components. Previously published peptidoglycan- and outer membrane-related gene mutants and strains with mutations in two genes (all5045 and alr0718) encoding class B penicillin-binding proteins isolated in this work were used. Our results show that filament length is affected in most cell envelope mutants, but the filaments of alr5045 and alr2270 gene mutants were particularly fragmented. All5045 is a dd-transpeptidase involved in peptidoglycan elongation during cell growth, and Alr2270 is an enzyme involved in the biosynthesis of lipid A, a key component of lipopolysaccharide. These results indicate that both components of the cell envelope, the murein sacculus and the outer membrane, influence filamentation. As deduced from the filament fragmentation phenotypes of their mutants, however, none of these elements is as important for filamentation as the septal protein SepJ.     

Ubiquitylation by Trim32 causes coupled loss of desmin, Z-bands, and thin filaments in muscle atrophy

During muscle atrophy, myofibrillar proteins are degraded in an ordered process in which MuRF1 catalyzes ubiquitylation of thick filament components (Cohen et al. 2009. J. Cell Biol. http://dx.doi.org/10.1083/jcb.200901052). Here, we show that another ubiquitin ligase, Trim32, ubiquitylates thin filament (actin, tropomyosin, troponins) and Z-band (α-actinin) components and promotes their degradation. Down-regulation of Trim32 during fasting reduced fiber atrophy and the rapid loss of thin filaments. Desmin filaments were proposed to maintain the integrity of thin filaments. Accordingly, we find that the rapid destruction of thin filament proteins upon fasting was accompanied by increased phosphorylation of desmin filaments, which promoted desmin ubiquitylation by Trim32 and degradation. Reducing Trim32 levels prevented the loss of both desmin and thin filament proteins. Furthermore, overexpression of an inhibitor of desmin polymerization induced disassembly of desmin filaments and destruction of thin filament components. Thus, during fasting, desmin phosphorylation increases and enhances Trim32-mediated degradation of the desmin cytoskeleton, which appears to facilitate the breakdown of Z-bands and thin filaments.     

Mos activates MAP kinase in mouse oocytes through two opposite pathways

Activation of mitogen-activated protein kinase (MAPK) in maturing mouse oocytes occurs after synthesis of Mos, a MAPKKK. To investigate whether Mos acts only through MEK1, we microinjected constitutively active forms of MEK1 (MEK1S218D/S222D referred herein as MEK*) and Raf (DeltaRaf) into mouse oocytes. In mos(-/-) oocytes, which do not activate MAPK during meiosis and do not arrest in metaphase II, MEK* and DeltaRaf did not rescue MAPK activation and metaphase II arrest, whereas Mos induced a complete rescue. MEK* and DeltaRaf induced cleavage arrest of two-cell blastomeres. They induced MAPK activation when protein phosphatases were inhibited by okadaic acid, suggesting that Mos may inhibit protein phosphatases. Finally, in mos(-/-) oocytes, MEK* induced the phosphorylation of Xp42(mapk)D324N, a mutant less sensitive to dephosphorylation, showing that a MAPK phosphatase activity is present in mouse oocytes. We demonstrate that active MAPKK or MAPKKK cannot substitute for Mos to activate MAPK in mouse oocytes. We also show that a phosphatase activity inactivates MAPK, and that Mos can overcome this inhibitory activity. Thus Mos activates MAPK through two opposite pathways: activation of MEK1 and inhibition of a phosphatase.     

Probing of the structural stability of vimentin and desmin-type intermediate filaments with Ca2+-activated proteinase, thrombin and lysine-specific endoproteinase Lys-C

Intermediate filaments (IFs) reconstituted from purified, delipidated vimentin and desmin as well as respective protofilaments were subjected to degradation by Ca2+-activated neutral thiol proteinase, thrombin and lysine-specific endoproteinase Lys-C, respectively. The breakdown products were analyzed by SDS-polyacrylamide gel electrophoresis and negative stain electron microscopy. While Ca2+-activated proteinase and thrombin caused rapid and complete degradation of IFs with kinetics not significantly different from those of the degradation of protofilaments, lysine-specific endoproteinase did not exert any electron microscopically detectable effect on filament structure. Although both types of subunit proteins were truncated at their non-alpha-helical, C-terminal polypeptides by this proteinase, they were still able to assemble into 10 nm filaments. Closer electron microscopic inspection of IFs treated with Ca2+-activated proteinase revealed numerous ruptures along the filaments already at very early stages of digestion. SDS-polyacrylamide gel electrophoresis of the processed filaments in conjunction with previous biochemical characterizations of the breakdown of protofilaments by Ca2+-activated proteinase showed that these inhomogeneities primarily arose from degradation of the arginine-rich, non-alpha-helical N-termini of the filament proteins. These findings demonstrate that, although the N-terminus of vimentin and desmin is essential for filament stability, it is still highly susceptible to proteolytic attack in particular and very likely to posttranslational modification in general. Such structural modifications of the N-termini of IF proteins might exert great influences on the intracellular distribution and molecular organization of IFs in various physiological and pathological conditions.     

Transposase-transposase interactions in MOS1 complexes: a biochemical approach

Transposases are proteins that have assumed the mobility of class II transposable elements. In order to map the interfaces involved in transposase-transposase interactions, we have taken advantage of 12 transposase mutants that impair mariner transposase-transposase interactions taking place during transposition. Our data indicate that transposase-transposase interactions regulating Mos1 transposition are sophisticated and result from (i) active MOS1 dimerization through the first HTH of the N-terminal domain, which leads to inverted terminal repeat (ITR) binding; (ii) inactive dimerization carried by part of the C-terminal domain, which prevents ITR binding; and (iii) oligomerization. Inactive dimers are nonpermissive in organizing complexes that produce ITR binding, but the interfaces (or interactions) supplied in this state could play a role in the various rearrangements needed during transposition. Oligomerization is probably not due to a specific MOS1 domain, but rather the result of nonspecific interactions resulting from incorrect folding of the protein. Our data also suggest that the MOS1 catalytic domain is a main actor in the overall organization of MOS1, thus playing a role in MOS1 oligomerization. Finally, we propose that MOS1 behaves as predicted by the pre-equilibrium existing model, whereby proteins are found to exist simultaneously in populations with diverse conformations, monomers and active and inactive dimers for MOS1. We were able to identify several MOS1 mutants that modify this pre-existing equilibrium. According to their properties, some of these mutants will be useful tools to break down the remaining gaps in our understanding of mariner transposition.     

A genome-wide screen in Saccharomyces cerevisiae reveals pathways affected by arsenic toxicity

We have used Saccharomyces cerevisiae to identify toxicologically important proteins and pathways involved in arsenic-induced toxicity and carcinogenicity in humans. We performed a systemic screen of the complete set of 4733 haploid S. cerevisiae single-gene-deletion mutants to identify those that have decreased or increased growth, relative to wild type, after exposure to sodium arsenite (NaAsO₂). IC₅₀ values for all mutants were determined to further validate our results. Ultimately we identified 248 mutants sensitive to arsenite and 5 mutants resistant to arsenite exposure. We analyzed the proteins corresponding to arsenite-sensitive mutants and determined that they belonged to functional categories that include protein binding, phosphate metabolism, vacuolar/lysosomal transport, protein targeting, sorting, and translocation, cell growth/morphogenesis, cell polarity and filament formation. Furthermore, these data were mapped onto a protein interactome to identify arsenite-toxicity-modulating networks. These networks are associated with the cytoskeleton, ubiquitination, histone acetylation and the MAPK signaling pathway. Our studies have potential implications for understanding toxicity and carcinogenesis in arsenic-induced human conditions, such as cancer and aging.     

Assembly of type IV neuronal intermediate filaments in nonneuronal cells in the absence of preexisting cytoplasmic intermediate filaments

We report here on the in vivo assembly of alpha-internexin, a type IV neuronal intermediate filament protein, in transfected cultured cells, comparing its assembly properties with those of the neurofilament triplet proteins (NF-L, NF-M, and NF-H). Like the neurofilament triplet proteins, alpha-internexin coassembles with vimentin into filaments. To study the assembly characteristics of these proteins in the absence of a preexisting filament network, transient transfection experiments were performed with a non-neuronal cell line lacking cytoplasmic intermediate filaments. The results showed that only alpha-internexin was able to self-assemble into extensive filamentous networks. In contrast, the neurofilament triplet proteins were incapable of homopolymeric assembly into filamentous arrays in vivo. NF-L coassembled with either NF-M or NF-H into filamentous structures in the transfected cells, but NF-M could not form filaments with NF-H. alpha- internexin could coassemble with each of the neurofilament triplet proteins in the transfected cells to form filaments. When all but 2 and 10 amino acid residues were removed from the tail domains of NF-L and NF-M, respectively, the resulting NF-L and NF-M deletion mutants retained the ability to coassemble with alpha-internexin into filamentous networks. These mutants were also capable of forming filaments with other wild-type neurofilament triplet protein subunits. These results suggest that the tail domains of NF-L and NF-M are dispensable for normal coassembly of each of these proteins with other type IV intermediate filament proteins to form filaments.     

Actin filaments in yeast are unstable in the absence of capping protein or fimbrin

Many actin-binding proteins affect filament assembly in vitro and localize with actin in vivo, but how their molecular actions contribute to filament assembly in vivo is not understood well. We report here that capping protein (CP) and fimbrin are both important for actin filament assembly in vivo in Saccharomyces cerevisiae, based on finding decreased actin filament assembly in CP and fimbrin mutants. We have also identified mutations in actin that enhance the CP phenotype and find that those mutants also have decreased actin filament assembly in vivo. In vitro, actin purified from some of these mutants is defective in polymerization or binding fimbrin. These findings support the conclusion that CP acts to stabilize actin filaments in vivo. This conclusion is particularly remarkable because it is the opposite of the conclusion drawn from recent studies in Dictyostelium (Hug, C., P.Y. Jay, I. Reddy, J.G. McNally, P.C. Bridgman, E.L. Elson, and J.A. Cooper. 1995. Cell. 81:591-600). In addition, we find that the unpolymerized pool of actin in yeast is very small relative to that found in higher cells, which suggests that actin filament assembly is less dynamic in yeast than higher cells.