α‐ and β‐Tubulin Phylogenies Support a Close Relationship Between the Microsporidia Brachiola algerae and Antonospora locustae

ABSTRACT. Microsporidia are a large and diverse group of intracellular parasites related to fungi. Much of our understanding of the relationships between microsporidia comes from phylogenies based on a single gene, the small subunit (SSU) rRNA, because only this gene has been sampled from diverse microsporidia. However, SSUrRNA trees are limited in their ability to resolve basal branches and some microsporidian affiliations are inconsistent between different analyses. Protein phylogenies have provided insight into relationships within specific groups of microsporidia, but have rarely been applied to the group as a whole. We have sequenced α‐ and β‐tubulins from microsporidia from three different subgroups, including representatives from what have previously been inferred to be the basal branches, allowing the broadest sampled protein‐based phylogenetic analysis to date. Although some relationships remain unresolved, many nodes uniting subgroups are strongly supported and consistent in both individual trees as well as a concatenate of both tubulins. One such relationship that was previously unclear is between Brachiola algerae and Antonospora locustae, and their close association with Encephalitozoon and Nosema. Also, an uncultivated microsporidian that infects cyclopoid copepods is shown to be related to Edhazardia aedis.     

Combined field efficacy of <Emphasis Type="Italic">Paranosema locustae</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> var. <Emphasis Type="Italic">acridum</Emphasis> for the control of sahelian grasshoppers

Field trials were conducted to evaluate the efficacy of wheat bran bait formulations of Paranosema locustae and Metarhizium anisopliae for controlling grasshoppers in southeast Niger. Treatments consisted of wheat bran baits mixed with M. anisopliae, P. locustae + M. anisopliae or with P. locustae spores and P. locustae + sugar. Oedaleus senegalensis, Pyrgomorpha cognata and Acrotylus blondeli were the predominant species at the time of application representing ca. 94% of the total population. Bran application was done when O. senegalensis (ca. 75% of the population) was at its early developmental stages, with first, second and third instars accounting for 64–85%. Grasshopper population reduction, P. locustae prevalence and level of infections in the predominant species were monitored. Manual application of P. locustae and M. anisopliae formulated in wheat bran has proven to induce consistent pathogen infection in grasshopper populations. Population density over the three weeks monitoring, typically decreased by 44.7 ± 6.9%, 52.8 ± 8.4%, 73.7 ± 5.5% and 89.1 ± 1.8% in P. locustae, P. locustae + sugar, M. anisopliae and P. locustae + M. anisopliae treated plots respectively. Paranosema locustae prevalence in surviving adult grasshoppers at 28 after application was 48.1 ± 2.3%, 28.9 ± 4.8% and 27.4 ± 3.7%, with infection level of 6.2 ± 0.8 × 10⁶, 2.3 ± 0.3 × 10⁴ and 2.1 ± 0.3 × 10³ spores mg⁻¹ host weight in O. senegalensis, A blondeli and P. cognate respectively. Other species that each accounted for <2% of the community, namely Aiolopus thalassinus, A. simulatrix, Acorypha glaucopsis, Acrotylus patruelis, Anacridium melanorhodon, Diabolocatantops axillaris, Kraussaria angulifera and Schistocerca gregaria were found to show sign of infection. The results from this study suggest that wheat bran application of M. anisopliae and P. locustae alone or in combination, targeting early instars grasshopper could be a valuable option in grasshopper control programs.     

Secretion of Antonospora (Paranosema) locustae Proteins into Infected Cells Suggests an Active Role of Microsporidia in the Control of Host Programs and Metabolic Processes

Molecular tools of the intracellular protozoan pathogens Apicomplexa and Kinetoplastida for manipulation of host cell machinery have been the focus of investigation for approximately two decades. Microsporidia, fungi-related microorganisms forming another large group of obligate intracellular parasites, are characterized by development in direct contact with host cytoplasm (the majority of species), strong minimization of cell machinery, and acquisition of unique transporters to exploit host metabolic system. All the aforementioned features are suggestive of the ability of microsporidia to modify host metabolic and regulatory pathways. Seven proteins of the microsporidium Antonospora (Paranosema) locustae with predicted signal peptides but without transmembrane domains were overexpressed in Escherichia coli. Western-blot analysis with antibodies against recombinant products showed secretion of parasite proteins from different functional categories into the infected host cell. Secretion of parasite hexokinase and α/β-hydrolase was confirmed by immunofluorescence microscopy. In addition, this method showed specific accumulation of A. locustae hexokinase in host nuclei. Expression of hexokinase, trehalase, and two leucine-rich repeat proteins without any exogenous signal peptide led to their secretion in the yeast Pichia pastoris. In contrast, α/β-hydrolase was not found in the culture medium, though a significant amount of this enzyme accumulated in the yeast membrane fraction. These results suggest that microsporidia possess a broad set of enzymes and regulatory proteins secreted into infected cells to control host metabolic processes and molecular programs.     

The Sec34/Sec35p complex,a Ypt1p effector required for retrograde intra-Golgi trafficking,interacts with Golgi SNAREs and COPI vesicle coat proteins

The Sec34/35 complex was identified as one of the evolutionarily conserved protein complexes that regulates a cis-Golgi step in intracellular vesicular transport. We have identified three new proteins that associate with Sec35p and Sec34p in yeast cytosol. Mutations in these Sec34/35 complex subunits result in defects in basic Golgi functions, including glycosylation of secretory proteins, protein sorting, and retention of Golgi resident proteins. Furthermore, the Sec34/35 complex interacts genetically and physically with the Rab protein Ypt1p, intra-Golgi SNARE molecules, as well as with Golgi vesicle coat complex COPI. We propose that the Sec34/35 protein complex acts as a tether that connects cis-Golgi membranes and COPI-coated, retrogradely targeted intra-Golgi vesicles.     

Carbon tetrachloride suppresses ER-Golgi transport by inhibiting COPII vesicle formation on the ER membrane in the RLC-16 hepatocyte cell line

Carbon tetrachloride (CCl₄) causes hepatotoxicity in mammals, with its hepatocytic metabolism producing radicals that attack the intracellular membrane system and destabilize intracellular vesicle transport. Inhibition of intracellular transport causes lipid droplet retention and abnormal protein distribution. The intracellular transport of synthesized lipids and proteins from the endoplasmic reticulum (ER) to the Golgi apparatus is performed by coat complex II (COPII) vesicle transport, but how CCl₄ inhibits COPII vesicle transport has not been elucidated. COPII vesicle formation on the ER membrane is initiated by the recruitment of Sar1 protein from the cytoplasm to the ER membrane, followed by that of the COPII coat constituent proteins (Sec23, Sec24, Sec13, and Sec31). In this study, we evaluated the effect of CCl₄ on COPII vesicle formation using the RLC-16 rat hepatocyte cell line. Our results showed that CCl₄ suppressed ER-Golgi transport in RLC-16 cells. Using a reconstituted system of rat liver tissue-derived cytoplasm and RLC-16 cell-derived ER membranes, CCl₄ treatment inhibited the recruitment of Sar1 and Sec13 from the cytosolic fraction to ER membranes. CCl₄-induced changes in the ER membrane accordingly inhibited the accumulation of COPII vesicle-coated constituent proteins on the ER membrane, as well as the formation of COPII vesicles, which suppressed lipid and protein transport between the ER and Golgi apparatus. Our data suggest that CCl₄ inhibits ER-Golgi intracellular transport by inhibiting COPII vesicle formation on the ER membrane in hepatocytes.     

COPI-independent Anterograde Transport: Cargo-selective ER to Golgi Protein Transport in Yeast COPI Mutants

The coatomer (COPI) complex mediates Golgi to ER recycling of membrane proteins containing a dilysine retrieval motif. However, COPI was initially characterized as an anterograde-acting coat complex. To investigate the direct and primary role(s) of COPI in ER/Golgi transport and in the secretory pathway in general, we used PCR-based mutagenesis to generate new temperature-conditional mutant alleles of one COPI gene in Saccharomyces cerevisiae, SEC21 (γ-COP). Unexpectedly, all of the new sec21 ts mutants exhibited striking, cargo-selective ER to Golgi transport defects. In these mutants, several proteins (i.e., CPY and α-factor) were completely blocked in the ER at nonpermissive temperature; however, other proteins (i.e., invertase and HSP150) in these and other COPI mutants were secreted normally. Nearly identical cargo-specific ER to Golgi transport defects were also induced by Brefeldin A. In contrast, all proteins tested required COPII (ER to Golgi coat complex), Sec18p (NSF), and Sec22p (v-SNARE) for ER to Golgi transport. Together, these data suggest that COPI plays a critical but indirect role in anterograde transport, perhaps by directing retrieval of transport factors required for packaging of certain cargo into ER to Golgi COPII vesicles. Interestingly, CPY–invertase hybrid proteins, like invertase but unlike CPY, escaped the sec21 ts mutant ER block, suggesting that packaging into COPII vesicles may be mediated by cis-acting sorting determinants in the cargo proteins themselves. These hybrid proteins were efficiently targeted to the vacuole, indicating that COPI is also not directly required for regulated Golgi to vacuole transport. Additionally, the sec21 mutants exhibited early Golgi-specific glycosylation defects and structural aberrations in early but not late Golgi compartments at nonpermissive temperature. Together, these studies demonstrate that although COPI plays an important and most likely direct role both in Golgi–ER retrieval and in maintenance/function of the cis-Golgi, COPI does not appear to be directly required for anterograde transport through the secretory pathway.     

Persistence of <Emphasis Type="Italic">Paranosema</Emphasis> (<Emphasis Type="Italic">Nosema</Emphasis>) <Emphasis Type="Italic">locustae</Emphasis> (Microsporidia: Nosematidae) among grasshopper (Orthoptera: Acrididae) populations in the Inner Mongolia Rangeland,China

Paranosema (Nosema) locustae Canning has persisted in grasshopper populations in the Inner Mongolia Rangeland since spore treatment was applied in 1993. In the treatment year, a 58.32–71.43% reduction of grasshoppers was recorded and 59.26–80.36% of surviving grasshoppers of various species were infected. In the years following application, prevalence of P. locustae varied from 13.65% to 60.71%, demonstrating that P. locustae remains in grasshopper populations for many years. Infection prevalence was more than 60.00% for Dasyhippus barbipes (F.-W.), Bryodema luctuosum luctuosoma (Stoll) and Angaracris barabensis (Pall.) in 1993 but was very variable, ranging from 20.00% to 60.71% over the subsequent nine years. Disease prevalence was 43.72–60.71% in 1993 and 1994 for Oedaleus asiaticus B.-Bienko and Myrmeleotettix palpalis (Zub.) but gradually declined to approximately 20%, where it remained. Persistence of P. locustae in grasshopper populations may be important in keeping densities below economic thresholds for a number of years after application. Handling Editor: Helen Roy.     

Predominant membrane localization is an essential feature of the bacterial signal recognition particle receptor

Background  

The signal recognition particle (SRP) receptor plays a vital role in co-translational protein targeting, because it connects the soluble SRP-ribosome-nascent chain complex (SRP-RNCs) to the membrane bound Sec translocon. The eukaryotic SRP receptor (SR) is a heterodimeric protein complex, consisting of two unrelated GTPases. The SRβ subunit is an integral membrane protein, which tethers the SRP-interacting SRα subunit permanently to the endoplasmic reticulum membrane. The prokaryotic SR lacks the SRβ subunit and consists of only the SRα homologue FtsY. Strikingly, although FtsY requires membrane contact for functionality, cell fractionation studies have localized FtsY predominantly to the cytosolic fraction of Escherichia coli. So far, the exact function of the soluble SR in E. coli is unknown, but it has been suggested that, in contrast to eukaryotes, the prokaryotic SR might bind SRP-RNCs already in the cytosol and only then initiates membrane targeting.     

The mammalian homolog of yeast Sec13p is enriched in the intermediate compartment and is essential for protein transport from the endoplasmic reticulum to the Golgi apparatus.

The role of COPII components in endoplasmic reticulum (ER)-Golgi transport, first identified in the yeast Saccharomyces cerevisiae, has yet to be fully characterized in higher eukaryotes. A human cDNA whose predicted amino acid sequence showed 70% similarity to the yeast Sec13p has previously been cloned. Antibodies raised against the human SEC13 protein (mSEC13) recognized a cellular protein of 35 kDa in both the soluble and membrane fractions. Like the yeast Sec13p, mSEC13 exist in the cytosol in both monomeric and higher-molecular-weight forms. Immunofluorescence microscopy localized mSEC13 to the characteristic spotty ER-Golgi intermediate compartment (ERGIC) in cells of all species examined, where it colocalized well with the KDEL receptor, an ERGIC marker, at 15 degrees C. Immunoelectron microscopy also localized mSEC13 to membrane structures close to the Golgi apparatus. mSEC13 is essential for ER-to-Golgi transport, since both the His6-tagged mSEC13 recombinant protein and the affinity-purified mSEC13 antibody inhibited the transport of restrictive temperature-arrested vesicular stomatitis virus G protein from the ER to the Golgi apparatus in a semi-intact cell assay. Moreover, cytosol immunodepleted of mSEC13 could no longer support ER-Golgi transport. Transport could be restored in a dose-dependent manner by a cytosol fraction enriched in the high-molecular-weight mSEC13 complex but not by a fraction enriched in either monomeric mSEC13 or recombinant mSEC13. As a putative component of the mammalian COPII complex, mSEC13 showed partially overlapping but mostly different properties in terms of localization, membrane recruitment, and dynamics compared to that of beta-COP, a component of the COPI complex.     

Carotenoid Accumulation and Carotenogenic Genes Expression During Two Types of Persimmon Fruit (<Emphasis Type="Italic">Diospyros kaki</Emphasis> L.) Development

Persimmon (Diospyros kaki L.), belonging to the Ebenaceae family, has been used not only as a fresh fruit, but also for many medicinal uses. Carotenoids are the main pigment in persimmon fruit, which contribute significantly to fruit color and nutritional quality due to their composition and content. In this study, fruit quality indices, carotenoid contents and expression of carotenogenic genes were analyzed in two types of persimmon fruit. The results demonstrated that there was a positive correlation between fruit color and the contents of main composition and total carotenoids. Carotenoid accumulation in persimmon fruit resulted from the interaction of carotenogenic genes, but the molecular mechanisms responsible for accumulation of carotenoids in two types of persimmon fruit had a few differences. As a complete unit, the relatively low expression level of phytoene synthase gene (DkPSY) in “Niuxinshi” resulted in low carotenoid contents or even under the detection limit at the early fruit developmental stages; but low carotenoid contents in “Nishimurawase” were due to the relatively low expression level of carotenogenic genes other than DkPSY. At the late fruit developmental stages, increased expression levels of DkPSY, phytoene desaturase gene and beta-carotene hydroxylase gene (DkBCH) induced elevated carotenoid contents; because all carotenogenic genes strongly expressed in “Nishimurawase”, a large amount of carotenoids were accumulated. In addition, β-cryptoxanthin was the main composition whose content increased with the fruit maturity changes, which was mainly because of DkBCH which might lead more conversion of β-carotene to β-cryptoxanthin.     

Functional characterization of the trans-membrane domain interactions of the Sec61 protein translocation complex beta-subunit

Background  

In eukaryotic cells co- and post-translational protein translocation is mediated by the trimeric Sec61 complex. Currently, the role of the Sec61 complex β-subunit in protein translocation is poorly understood. We have shown previously that in Saccharomyces cerevisiae the trans-membrane domain alone is sufficient for the function of the β-subunit Sbh1p in co-translational protein translocation. In addition, Sbh1p co-purifies not only with the protein translocation channel subunits Sec61p and Sss1p, but also with the reticulon family protein Rtn1p.     

Scission of COPI and COPII Vesicles Is Independent of GTP Hydrolysis

Intracellular transport and maintenance of the endomembrane system in eukaryotes depends on formation and fusion of vesicular carriers. A seeming discrepancy exists in the literature about the basic mechanism in the scission of transport vesicles that depend on GTP‐binding proteins. Some reports describe that the scission of COP‐coated vesicles is dependent on GTP hydrolysis, whereas others found that GTP hydrolysis is not required. In order to investigate this pivotal mechanism in vesicle formation, we analyzed formation of COPI‐ and COPII‐coated vesicles utilizing semi‐intact cells. The small GTPases Sar1 and Arf1 together with their corresponding coat proteins, the Sec23/24 and Sec13/31 complexes for COPII and coatomer for COPI vesicles were required and sufficient to drive vesicle formation. Both types of vesicles were efficiently generated when GTP hydrolysis was blocked either by utilizing the poorly hydrolyzable GTP analogs GTPγS and GMP‐PNP, or with constitutively active mutants of the small GTPases. Thus, GTP hydrolysis is not required for the formation and release of COP vesicles.     

Bacterial catalase in the microsporidian Nosema locustae: implications for microsporidian metabolism and genome evolution

Microsporidia constitute a group of extremely specialized intracellular parasites that infect virtually all animals. They are highly derived, reduced fungi that lack several features typical of other eukaryotes, including canonical mitochondria, flagella, and peroxisomes. Consistent with the absence of peroxisomes in microsporidia, the recently completed genome of the microsporidian Encephalitozoon cuniculi lacks a gene for catalase, the major enzymatic marker for the organelle. We show, however, that the genome of the microsporidian Nosema locustae, in contrast to that of E. cuniculi, encodes a group II large-subunit catalase. Surprisingly, phylogenetic analyses indicate that the N. locustae catalase is not specifically related to fungal homologs, as one would expect, but is instead closely related to proteobacterial sequences. This finding indicates that the N. locustae catalase is derived by lateral gene transfer from a bacterium. The catalase gene is adjacent to a large region of the genome that appears to be far less compact than is typical of microsporidian genomes, a characteristic which may make this region more amenable to the insertion of foreign genes. The N. locustae catalase gene is expressed in spores, and the protein is detectable by Western blotting. This type of catalase is a particularly robust enzyme that has been shown to function in dormant cells, indicating that the N. locustae catalase may play some functional role in the spore. There is no evidence that the N. locustae catalase functions in a cryptic peroxisome.     

Interactions of two insect pathogens, Paranosema locustae (Protista: Microsporidia) and Metarhizium acridum (Fungi: Hypocreales), during a mixed infection of Locusta migratoria (Insecta: Orthoptera) nymphs

Locusta migratoria nymphs were fed Paranosema locustae spores and/or surface-treated with Metarhizium acridum 3 (assay 1), 6 (assay 2) or 9 days (assay 3) post microsporidia application (p.m.a.). These three dates corresponded to the key phases of P. locustae development: (a) mass proliferation, (b) transition to sporogenesis and (c) onset of spore maturation, respectively. As a result, locust mortality due to mixed treatment increased slower, equally and faster, as compared to mortality expected from the combination of two pathogens in assays 1-3, respectively. However, a statistically significant difference in survival times was observed only in assay 3, indicating that only at the phase of spore maturation microsporidia drastically increase locust susceptibility to fungal infection. Analysis of perished nymphs showed that fungal treatment 3 days p.m.a. impeded development of microsporidia. Fungal sporulation on locust cadavers was not affected by co-occurring microsporidiosis.     

Differential susceptibility of two locust pests to the microsporidium Paranosema (Antonospora) locustae at suboptimal rearing temperature

Paranosema (Antonospora) locustae (Canning) is a microsporidium with an extensive host range including >100 reported insect hosts from the order Orthoptera. The susceptibility of two species of locusts (Orthoptera: Acrididae) – the migratory locust, Locusta migratoria subsp. migratorioides (Fairmaire & Reiche), and the desert locust, Schistocerca gregaria Forsskål – to P. locustae was compared under laboratory conditions at a decreased temperature of 27 °C to enhance susceptibility to infection. Locusta migratoria was found highly vulnerable as infection percentages exceeded 70% at 10⁴, 10⁵, and 10⁶ spores per insect, and mortality increased with increasing dosage. Conversely, P. locustae spores were not found in S. gregaria throughout the experiment. Only at 10⁷ spores per insect, as many as 20% of S. gregaria were infected. This suggests that the desert locust is resistant to P. locustae infection, as opposed to the migratory locust. The laboratory models of these parasite–host systems may be useful for elucidating mechanisms of insect immunity to microsporidia.     

Effect of entomocidal proteins from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> on ion permeability of apical membranes of <Emphasis Type="Italic">Tenebrio molitor</Emphasis> larvae gut epithelium

Effects of entomocidal Cry-type proteins, δ-endotoxins Cry3A and Cry11A produced by Bacillus thuringiensis, on ion permeability of the apical membranes of intestinal epithelium from Tenebrio molitor larvae midgut were studied. Using potential-sensitive dyes safranine O and oxonol VI and δpH indicator acridine orange, it was shown that placing brush border membrane vesicles (BBMV) (loaded with Mg²⁺ during their preparation) into a salt-free buffer medium resulted in spontaneous generation of transmembrane electric potential on the vesicular membrane (negative inside the vesicles) accompanied by acidification of the aqueous phase inside the vesicles. The generation of transmembrane ion gradients on the vesicular membrane was a result of an electrogenic efflux of Mg²⁺ from the vesicles as shown by abolishing of the membrane potential by such agents as MgSO₄ or CaCl₂ in centimolar concentrations, a highly lipophilic cation tetraphenylphosphonium, and some blockers of cell membrane Ca²⁺-channels in submillimolar concentrations. A passive generation of membrane potential on the vesicular membrane (but positive inside the vesicles) was also observed upon addition of centimolar concentrations of K₂SO₄. Addition of δ-endotoxins Cry3A and Cry11A to the vesicle suspension in a salt-free buffer medium or in the same medium supplemented with centimolar concentrations of K₂SO₄ exerted a pronounced hyperpolarization of the vesicular membrane. This hyperpolarization was sensitive to the same agents, which abolished the membrane potential generation in the absence of δ-endotoxin. It is concluded that Cry proteins induced in BBMV from T. molitor opening pores or ion channels, which were considerably more permeable for alkaline- and alkaline-earth metal cations than for the accompanying anions.     

Biosynthesis of medium-chain-length poly(hydroxyalkanoates) with altered composition by mutant hybrid PHA synthases

Pseudomonas resinovorans harbors two isogenic poly(hydroxyalkanoates) (PHAs) synthase genes (phaC1 _Pre , phaC2 _Pre ) responsible for the production of intracellular medium-chain-length (mcl-)PHAs. Sequence analysis showed that the putative gene-products of these genes contain a conserved α/β-hydrolase fold in the carboxy-terminal half of the proteins. Hybrid genes pha7 and pha8 were constructed by exchanging the α/β-hydrolase-fold coding portions of phaC1 _Pre and phaC2 _Pre at the 3′ terminal. When grown with decanoate as carbon source, the pha7- or pha8-transformed Escherichia coli LS1298 produced PHAs containing 73–75% β-hydroxydecanoate (β-HD) and 25–27% β-hydroxyoctanoate (β-HO). Deletion mutants, Δpha7 and Δpha8, were isolated during the PCR-based construction of pha7 and pha8, respectively. Cells harboring these mutants produced PHAs containing 55–60 mol% β-HD and 40–45 mol% β-HO. These results demonstrate the feasibility of generating active hybrid mcl-PHA synthase genes and their mutants with the potential of producing polymers having a varied repeat-unit composition.     

Polarized membrane distribution of potassium-dependent ion pumps in epithelial cells: Different roles of the <Emphasis Type="Italic">N</Emphasis>-glycans of their <Emphasis Type="Italic">β</Emphasis> subunits

The Na,K-ATPases and the H,K-ATPases are two potassium-dependent homologous heterodimeric P₂-type pumps that catalyze active transport of Na⁺ in exchange for K⁺ (Na,K-ATPase) or H⁺ in exchange for K⁺ (H,K-ATPase). The ubiquitous Na,K-ATPase maintains intracellular ion balance and membrane potential. The gastric H,K-ATPase is responsible for acid secretion by the parietal cell of the stomach. Both pumps consist of a catalytic α-subunit and a glycosylated β-subunit that is obligatory for normal pump maturation and trafficking. Individual N-glycans linked to the β-subunits of the Na,K-ATPase and H,K-ATPase are important for stable membrane integration of their respective α subunits, folding, stability, subunit assembly, and enzymatic activity of the pumps. They are also essential for the quality control of unassembled β-subunits that results in either the exit of the subunits from the ER or their ER retention and subsequent degradation. Overall, the importance of N-glycans for the␣maturation and quality control of the H,K-ATPase is greater than that of the Na,K-ATPase. The roles of individual N-glycans of the β-subunits in the post-ER trafficking, membrane targeting and plasma membrane retention of the Na,K-ATPase and H,K-ATPase are different. The Na,K-ATPase β ₁-subunit is the major β-subunit isoform in cells with lateral location of the pump. All three N-glycans of the Na,K-ATPase β ₁-subunit are important for the lateral membrane retention of the pump due to glycan-mediated interaction between the β ₁-subunits of the two neighboring cells in the cell monolayer and cytosolic linkage of the α-subunit to the cytoskeleton. This intercellular β ₁–β ₁ interaction is also important for formation of cell–cell contacts. In contrast, the N-glycans unique to the Na,K-ATPase β ₂-subunit,which has up to eight N-glycosylation sites, contain apical sorting information. This is consistent with the apical location of the Na,K-ATPase in normal and malignant epithelial cells with high abundance of the β ₂-subunit. Similarly, all seven N-glycans of the gastric H,K-ATPase β-subunit determine apical sorting of this subunit. Supported in part by NIH grants DK46917, DK58333, D53642, and USVA     

The coatomer-interacting protein Dsl1p is required for Golgi-to-endoplasmic reticulum retrieval in yeast

Sec22p is an endoplasmic reticulum (ER)-Golgi v-SNARE protein whose retrieval from the Golgi compartment to the endoplasmic reticulum (ER) is mediated by COPI vesicles. Whether Sec22p exhibits its primary role at the ER or the Golgi apparatus is still a matter of debate. To determine the role of Sec22p in intracellular transport more precisely, we performed a synthetic lethality screen. We isolated mutant yeast strains in which SEC22 gene function, which in a wild type strain background is non-essential for cell viability, has become essential. In this way a novel temperature-sensitive mutant allele, dsl1-22, of the essential gene DSL1 was obtained. The dsl1-22 mutation causes severe defects in Golgi-to-ER retrieval of ER-resident SNARE proteins and integral membrane proteins harboring a C-terminal KKXX retrieval motif, as well as of the soluble ER protein BiP/Kar2p, which utilizes the HDEL receptor, Erd2p, for its recycling to the ER. DSL1 interacts genetically with mutations that affect components of the Golgi-to-ER recycling machinery, namely sec20-1, tip20-5, and COPI-encoding genes. Furthermore, we demonstrate that Dsl1p is a peripheral membrane protein, which in vitro specifically binds to coatomer, the major component of the protein coat of COPI vesicles.