Yeast and human Ysl2p/hMon2 interact with Gga adaptors and mediate their subcellular distribution

The Gga proteins represent a family of ubiquitously expressed clathrin adaptors engaged in vesicle budding at the tubular endosomal network/trans Golgi network. Their membrane recruitment is commonly thought to involve interactions with Arf and signals in cargo through the so-called VHS domain. For yeast Gga proteins, however, partners binding to its VHS domain have remained elusive and Gga localization does not absolutely depend on Arf. Here, we demonstrate that yeast Gga recruitment relies on a network of interactions between the scaffold Ysl2p/Mon2p, the small GTPase Arl1p, and the flippase Neo1p. Deletion of either YSL2 or ARL1 causes mislocalization of Gga2p, whereas a neo1-69 mutant accumulates Gga2p on aberrant structures. Remarkably, Ysl2p directly interacts with human and yeast Ggas through the VHS domain, and binding to Gga proteins is also found for the human Ysl2p orthologue hMon2. Thus, Ysl2p represents an essential, evolutionarily conserved member of a network controlling direct binding and membrane docking of Ggas. Because activated Arl1p is part of the network that binds Gga2p, Arf and Arf-like GTPases may interact in a regulatory cascade.     

Molecular interactions of yeast Neo1p, an essential member of the Drs2 family of aminophospholipid translocases, and its role in membrane trafficking within the endomembrane system

Neo1p is an essential yeast member of the highly conserved Drs2 family of P-type ATPases with proposed aminophospholipid translocase activity. Here we present evidence that Neo1p localizes to endosomes and Golgi elements. In agreement with that finding, the temperature-sensitive neo1-37 and neo1-69 mutants exhibit defects in receptor-mediated endocytosis, vacuole biogenesis, and vacuolar protein sorting. Furthermore, neo1 mutants accumulate aberrantly shaped membranous structures most likely derived from vacuoles and the endosomal/Golgi system. At permissive temperatures, HA-Neo1-69p, like wild-type Neo1p, is stable and associates with endosomes. In contrast, HA-Neo1-37p is rapidly degraded and is predominantly retained within the endoplasmic reticulum (ER). Thus, the two neo1 alleles affect the stability and localization of the mutant polypeptides in different ways. A C-terminally truncated and a C-terminally epitope-tagged version of Neo1p are nonfunctional and also mislocalize to the ER. In agreement with a role within the endomembrane system, Neo1p exhibits genetic and physical interactions with Ysl2p, a potential guanine nucleotide exchange factor for Arl1p. Interestingly, deletion of ARL1 rescues the temperature sensitivity of neo1-37 and neo1-69. We demonstrate that Arl1p in its myristoylated and GTP-bound form is responsible for the inhibitory effect. Thus, Neo1p, Ysl2p, and Arl1p represent three proteins that collaborate in membrane trafficking within the endosomal/Golgi system.     

Yeast Dop1 is required for glycosyltransferase retrieval from the trans-Golgi network

BackgroundGlycosyltransferases are type II membrane proteins that are responsible for glycan modification of proteins and lipids, and localize to distinct cisternae in the Golgi apparatus. During cisternal maturation, retrograde trafficking helps maintain the steady-state localization of these enzymes in the sub-compartments of the Golgi.MethodsTo understand how glycosyltransferases are recycled in the late Golgi complex, we searched for genes that are essential for budding yeast cell growth and that encode proteins localized in endosomes and in the Golgi. We specifically analyzed the roles of Dop1 and its binding partner Neo1 in retaining Golgi-resident glycosyltransferases, in the late Golgi complex.ResultsDop1 primarily localized to younger compartments of the trans-Golgi network (TGN) and seemed to cycle within the TGN. In contrast, Neo1, a P4-ATPase that interacts with Dop1, localized to the TGN. Abolition of DOP1 expression led to defects in the FM4-64 endocytic pathway. Dop1 and Neo1 were required for correct glycosylation of invertase, a secretory protein, at the Golgi. In DOP1-shutdown cells, Och1, a mannosyltransferase that is typically located in the cis-Golgi, mislocalized to the TGN. In addition, the function of multiple glycosyltransferases required for N- and O-glycosylation were impaired in DOP1-shutdown cells.ConclusionsOur results indicate that Dop1 is involved in vesicular transport at the TGN, and is critical for retrieving glycosyltransferases from the TGN to the Golgi in yeast.General significanceGolgi-resident glycosyltransferases recycling from the TGN to the Golgi is dependent on Dop1 and the P4-ATPase Neo1.     

A role for septin 2 in Drp1‐mediated mitochondrial fission

Mitochondria are essential eukaryotic organelles often forming intricate networks. The overall network morphology is determined by mitochondrial fusion and fission. Among the multiple mechanisms that appear to regulate mitochondrial fission, the ER and actin have recently been shown to play an important role by mediating mitochondrial constriction and promoting the action of a key fission factor, the dynamin‐like protein Drp1. Here, we report that the cytoskeletal component septin 2 is involved in Drp1‐dependent mitochondrial fission in mammalian cells. Septin 2 localizes to a subset of mitochondrial constrictions and directly binds Drp1, as shown by immunoprecipitation of the endogenous proteins and by pulldown assays with recombinant proteins. Depletion of septin 2 reduces Drp1 recruitment to mitochondria and results in hyperfused mitochondria and delayed FCCP‐induced fission. Strikingly, septin depletion also affects mitochondrial morphology in Caenorhabditis elegans, strongly suggesting that the role of septins in mitochondrial dynamics is evolutionarily conserved.     

The Sec1/Munc18 Protein Groove Plays a Conserved Role in Interaction with Sec9p/SNAP‐25

The Sec1/Munc18 (SM) proteins constitute a conserved family with essential functions in SNARE‐mediated membrane fusion. Recently, a new protein–protein interaction site in Sec1p, designated the groove, was proposed. Here, we show that a sec1 groove mutant yeast strain, sec1(w24), displays temperature‐sensitive growth and secretion defects. The yeast Sec1p and mammalian Munc18‐1 grooves were shown to play an important role in the interaction with the SNAREs Sec9p and SNAP‐25b, respectively. Incubation of SNAP‐25b with the Munc18‐1 groove mutant resulted in a lag in the kinetics of SNARE complex assembly in vitro when compared with wild‐type Munc18‐1. The SNARE regulator SRO7 was identified as a multicopy suppressor of sec1(w24) groove mutant and an intact Sec1p groove was required for the plasma membrane targeting of Sro7p–SNARE complexes. Simultaneous inactivation of Sec1p groove and SRO7 resulted in reduced levels of exocytic SNARE complexes. Our results identify the groove as a conserved interaction surface in SM proteins. The results indicate that this structural element is important for interactions with Sec9p/SNAP‐25 and participates, in concert with Sro7p, in the initial steps of SNARE complex assembly.      

HOPS prevents the disassembly of trans‐SNARE complexes by Sec17p/Sec18p during membrane fusion

SNARE‐dependent membrane fusion requires the disassembly of cis‐SNARE complexes (formed by SNAREs anchored to one membrane) followed by the assembly of trans‐SNARE complexes (SNAREs anchored to two apposed membranes). Although SNARE complex disassembly and assembly might be thought to be opposing reactions, the proteins promoting disassembly (Sec17p/Sec18p) and assembly (the HOPS complex) work synergistically to support fusion. We now report that trans‐SNARE complexes formed during vacuole fusion are largely associated with Sec17p. Using a reconstituted proteoliposome fusion system, we show that trans‐SNARE complex, like cis‐SNARE complex, is sensitive to Sec17p/Sec18p mediated disassembly. Strikingly, HOPS inhibits the disassembly of SNARE complexes in the trans‐, but not in the cis‐, configuration. This selective HOPS preservation of trans‐SNARE complexes requires HOPS:SNARE recognition and is lost when the apposed bilayers are dissolved in Triton X‐100; it is also observed during fusion of isolated vacuoles. HOPS thus directs the Sec17p/Sec18p chaperone system to maximize functional trans‐SNARE complex for membrane fusion, a new role of tethering factors during membrane traffic.     

Molecular analysis of the prokaryotic ubiquitin‐like protein (Pup) conjugation pathway in Mycobacterium tuberculosis

Proteins targeted for degradation by the Mycobacterium proteasome are post‐translationally tagged with prokaryotic ubiquitin‐like protein (Pup), an intrinsically disordered protein of 64 residues. In a process termed ‘pupylation’, Pup is synthesized with a terminal glutamine, which is deamidated to glutamate by Dop (deamidase of Pup) prior to attachment to substrate lysines by proteasome accessory factor A (PafA). Importantly, PafA was previously shown to be essential to cause lethal infections by Mycobacterium tuberculosis (Mtb) in mice. In this study we show that Dop, like PafA, is required for the full virulence of Mtb. Additionally, we show that Dop is not only involved in the deamidation of Pup, but also needed to maintain wild‐type steady state levels of pupylated proteins in Mtb. Finally, using structural models and site‐directed mutagenesis our data suggest that Dop and PafA are members of the glutamine synthetase fold family of proteins.     

Topology of the microsomal glycerol‐3‐phosphate acyltransferase Gpt2p/Gat1p of Saccharomyces cerevisiae

All glycerophospholipids are made from phosphatidic acid, which, according to the traditional view, is generated at the cytosolic surface of the ER. In yeast, phosphatidic acid is synthesized de novo by two acyl‐CoA‐dependent acylation reactions. The first is catalysed by one of the two homologous glycerol‐3‐phosphate acyltransferases Gpt2p/Gat1p and Sct1p/Gat2p, the second by one of the two 1‐acyl‐sn‐glycerol‐3‐phosphate acyltransferases Slc1p and Ale1p/Slc4p. To study the biogenesis and topology of Gpt2p we observed the location of dual topology reporters inserted after various transmembrane helices. Moreover, using microsomes, we probed the accessibility of natural and substituted cysteine residues to a membrane impermeant alkylating agent and tested the protease sensitivity of various epitope tags inserted into Gpt2p. Finally, we assayed the sensitivity of the acyltransferase activity to membrane impermeant agents targeting lysine residues. By all these criteria we find that the most conserved motifs of Gpt2p and its functionally relevant lysines are oriented towards the ER lumen. Thus, the first step in biosynthesis of phosphatidic acid in yeast seems to occur in the ER lumen and substrates may have to cross the ER membrane.     

Dop functions as a depupylase in the prokaryotic ubiquitin‐like modification pathway

Post‐translational modification of proteins with prokaryotic ubiquitin‐like protein (Pup) is the bacterial equivalent of ubiquitination in eukaryotes. Mycobacterial pupylation is a two‐step process in which the carboxy‐terminal glutamine of Pup is first deamidated by Dop (deamidase of Pup) before ligation of the generated γ‐carboxylate to substrate lysines by the Pup ligase PafA. In this study, we identify a new feature of the pupylation system by demonstrating that Dop also acts as a depupylase in the Pup proteasome system in vivo and in vitro. Dop removes Pup from substrates by specific cleavage of the isopeptide bond. Depupylation can be enhanced by the unfolding activity of the mycobacterial proteasomal ATPase Mpa.     

Adaptor Aly and co‐adaptor Thoc5 function in the Tap‐p15‐mediated nuclear export of HSP70 mRNA

In metazoans, nuclear export of bulk mRNA is mediated by Tap‐p15, a conserved heterodimeric export receptor that cooperates with adaptor RNA‐binding proteins. In this article, we show that Thoc5, a subunit of the mammalian TREX complex, binds to a distinct surface on the middle (Ntf2‐like) domain of Tap. Notably, adaptor protein Aly and Thoc5 can simultaneously bind to non‐overlapping binding sites on Tap‐p15. In vivo, Thoc5 was not required for bulk mRNA export. However, nuclear export of HSP70 mRNA depends on both Thoc5 and Aly. Consistent with a function as a specific export adaptor, Thoc5 exhibits in vitro RNA‐binding activity and is associated with HSP70 mRNPs in vivo as a component of the stable THO complex. Thus, through the combinatorial use of an adaptor (e.g., Aly) and co‐adapter (e.g., Thoc5), Tap‐p15 could function as an export receptor for different classes of mRNAs.     

Arabidopsis thaliana FLA4 functions as a glycan‐stabilized soluble factor via its carboxy‐proximal Fasciclin 1 domain

Fasciclin‐like arabinogalactan proteins (FLAs) are involved in numerous important functions in plants but the relevance of their complex structure to physiological function and cellular fate is unresolved. Using a fully functional fluorescent version of Arabidopsis thaliana FLA4 we show that this protein is localized at the plasma membrane as well as in endosomes and soluble in the apoplast. FLA4 is likely to be GPI‐anchored, is highly N‐glycosylated and carries two O‐glycan epitopes previously associated with arabinogalactan proteins. The activity of FLA4 was resistant against deletion of the amino‐proximal fasciclin 1 domain and was unaffected by removal of the GPI‐modification signal, a highly conserved N‐glycan or the deletion of predicted O‐glycosylation sites. Nonetheless these structural changes dramatically decreased endoplasmic reticulum (ER)‐exit and plasma membrane localization of FLA4, with N‐glycosylation acting at the level of ER‐exit and O‐glycosylation influencing post‐secretory fate. We show that FLA4 acts predominantly by molecular interactions involving its carboxy‐proximal fasciclin 1 domain and that its amino‐proximal fasciclin 1 domain is required for stabilization of plasma membrane localization. FLA4 functions as a soluble glycoprotein via its carboxy‐proximal Fas1 domain and its normal cellular trafficking depends on N‐ and O‐glycosylation.     

The GmFWL1 (FW2‐2‐like) nodulation gene encodes a plasma membrane microdomain‐associated protein

The soybean gene GmFWL1 (FW2‐2‐like1) belongs to a plant‐specific family that includes the tomato FW2‐2 and the maize CNR1 genes, two regulators of plant development. In soybean, GmFWL1 is specifically expressed in root hair cells in response to rhizobia and in nodules. Silencing of GmFWL1 expression significantly reduced nodule numbers supporting its role during soybean nodulation. While the biological role of GmFWL1 has been described, its molecular function and, more generally, the molecular function of plant FW2‐2‐like proteins is unknown. In this study, we characterized the role of GmFWL1 as a membrane microdomain‐associated protein. Specifically, using biochemical, molecular and cellular methods, our data show that GmFWL1 interacts with various proteins associated with membrane microdomains such as remorin, prohibitins and flotillins. Additionally, comparative genomics revealed that GmFWL1 interacts with GmFLOT2/4 (FLOTILLIN2/4), the soybean ortholog to Medicago truncatula FLOTILLIN4, a major regulator of the M. truncatula nodulation process. We also observed that, similarly to MtFLOT4 and GmFLOT2/4, GmFWL1 was localized at the tip of the soybean root hair cells in response to rhizobial inoculation supporting the early function of GmFWL1 in the rhizobium infection process.     

The Skp1‐like protein SSK1 is required for cross‐pollen compatibility in S‐RNase‐based self‐incompatibility

The self‐incompatibility (SI) response occurs widely in flowering plants as a means of preventing self‐fertilization. In these self/non‐self discrimination systems, plant pistils reject self or genetically related pollen. In the Solanaceae, Plantaginaceae and Rosaceae, pistil‐secreted S‐RNases enter the pollen tube and function as cytotoxins to specifically arrest self‐pollen tube growth. Recent studies have revealed that the S‐locus F‐box (SLF) protein controls the pollen expression of SI in these families. However, the precise role of SLF remains largely unknown. Here we report that PhSSK1 (Petunia hybrida SLF‐interacting Skp1‐like1), an equivalent of AhSSK1 of Antirrhinum hispanicum, is expressed specifically in pollen and acts as an adaptor in an SCF(Skp1‐Cullin1‐F‐box)^SLF complex, indicating that this pollen‐specific SSK1‐SLF interaction occurs in both Petunia and Antirrhinum, two species from the Solanaceae and Plantaginaceae, respectively. Substantial reduction of PhSSK1 in pollen reduced cross‐pollen compatibility (CPC) in the S‐RNase‐based SI response, suggesting that the pollen S determinant contributes to inhibiting rather than protecting the S‐RNase activity, at least in solanaceous plants. Furthermore, our results provide an example that a specific Skp1‐like protein other than the known conserved ones can be recruited into a canonical SCF complex as an adaptor.     

Analysis of membrane–protein complexes of the marine sulfate reducer Desulfobacula toluolica Tol2 by 1D blue native‐PAGE complexome profiling and 2D blue native‐/SDS‐PAGE

Sulfate‐reducing bacteria (SRB) obtain energy from cytoplasmic reduction of sulfate to sulfide involving APS‐reductase (AprAB) and dissimilatory sulfite reductase (DsrAB). These enzymes are predicted to obtain electrons from membrane redox complexes, i.e. the quinone‐interacting membrane‐bound oxidoreductase (QmoABC) and DsrMKJOP complexes. In addition to these conserved complexes, the genomes of SRB encode a large number of other (predicted) membrane redox complexes, the function and actual formation of which is unknown. This study reports the establishment of 1D Blue Native‐PAGE complexome profiling and 2D BN‐/SDS‐PAGE for analysis of the membrane protein complexome of the marine sulfate reducer Desulfobacula toluolica Tol2. Analysis of normalized score profiles of >800 proteins in combination with hierarchical clustering and identification of 2D BN‐/SDS‐PAGE separated spots demonstrated separation of membrane complexes in their native form, e.g. ATP synthase. In addition to the QmoABC and DsrMKJOP complexes, other complexes were detected that constitute the basic membrane complexome of D. toluolica Tol2, e.g. transport proteins (e.g. sodium/sulfate symporters) or redox complexes involved in Na⁺‐based bioenergetics (RnfABCDEG). Notably, size estimation indicates dimer and quadruple formation of the DsrMKJOP complex in vivo. Furthermore, cluster analysis suggests interaction of this complex with a rhodanese‐like protein (Tol2_C05230) possibly representing a periplasmic electron transfer partner for DsrMKJOP.     

Proteomic analysis of irregular,bullet‐shaped magnetosomes in the sulphate‐reducing magnetotactic bacterium Desulfovibrio magneticus RS‐1

Recent molecular studies on magnetotactic bacteria have identified a number of proteins associated with bacterial magnetites (magnetosomes) and elucidated their importance in magnetite biomineralisation. However, these analyses were limited to magnetotactic bacterial strains belonging to the α‐subclass of Proteobacteria. We performed a proteomic analysis of magnetosome membrane proteins in Desulfovibrio magneticus strain RS‐1, which is phylogenetically classified as a member of the δ‐Proteobacteria. In the analysis, the identified proteins were classified based on their putative functions and compared with the proteins from the other magnetotactic bacteria, Magnetospirillum magneticum AMB‐1 and M. gryphiswaldense MSR‐1. Three magnetosome‐specific proteins, MamA (Mms24), MamK, and MamM, were identified in strains RS‐1, AMB‐1, and MSR‐1. Furthermore, genes encoding ten magnetosome membrane proteins, including novel proteins, were assigned to a putative magnetosome island that contains subsets of genes essential for magnetosome formation. The collagen‐like protein and putative iron‐binding proteins, which are considered to play key roles in magnetite crystal formation, were identified as specific proteins in strain RS‐1. Furthermore, genes encoding two homologous proteins of Magnetococcus MC‐1 were assigned to a cryptic plasmid of strain RS‐1. The newly identified magnetosome membrane proteins might contribute to the formation of the unique irregular, bullet‐shaped crystals in this microorganism.     

Rutin treats myocardial damage caused by pirarubicin via regulating miR‐22‐5p‐regulated RAP1/ERK signaling pathway

Our experiments have previously demonstrated that rutin (RUT) can improve myocardial damage caused by pirarubicin (THP). However, the underlying molecular mechanisms remain uncertain. In this study, we developed an microRNA (miRNA) chip by replicating the rat model of THP‐induced myocardial injury and identified miR‐22‐5p and the RAP1‐member of RAS oncogene family/extracellular regulated protein kinases (RAP1/ERK) signaling pathway as an object of study. Also, in vivo experiments demonstrated that THP caused abnormal changes in the electrocardiogram, cardiac function, and histomorphology in rats (P < .01). THP also reduces the expression of miR‐22‐5p (P < .01) and increases the levels of RAP1/ERK signaling pathway‐related proteins (P < .01, P < .05). RUT significantly improved THP‐induced myocardial damage (P < .01), increased the expression of miR‐22‐5p (P < .01), and decreased the levels of RAP1/ERK signaling pathway‐related proteins (P < .01, P < .05). In vitro studies confirmed that Rap1a is one of the target genes of miR‐22‐5p. miR‐22‐5p overexpression in cardiomyocytes can affect the RAP1/ERK pathway and reduce reactive oxygen species production and cardiomyocyte apoptosis caused by THP (P < .01), which is consistent with the effect of RUT. Our results indicate that RUT treats THP‐induced myocardial damage, which may be achieved by upregulating miR‐22‐5p, causing changes in its target gene Rap1a and the RAP1/ERK pathway.