Trafficking and release of Leishmania metacyclic HASPB on macrophage invasion

Proteins of the Leishmania hydrophilic acylated surface protein B (HASPB) family are only expressed in infective parasites (both extra- and intracellular stages) and, together with the peripheral membrane protein SHERP (small hydrophilic endoplasmic reticulum-associated protein), are essential for parasite differentiation (metacyclogenesis) in the sand fly vector. HASPB is a 'non-classically' secreted protein, requiring N-terminal acylation for trafficking to and exposure on the plasma membrane. Here, we use live cell imaging methods to further explore this pathway to the membrane and flagellum. Unlike HASPB trafficking in transfected mammalian cells, we find no evidence for a phosphorylation-regulated recycling pathway in metacyclic parasites. Once at the plasma membrane, HASPB18-GFP (green fluorescent protein) can undergo bidirectional movement within the inner leaflet of the membrane and on the flagellum. Transfer of fluorescent protein between the flagellum and the plasma membrane is compromised, however, suggesting the presence of a diffusion barrier at the base of the Leishmania flagellum. Full-length HASPB is released from the metacyclic parasite surface on to macrophages during phagocytosis but while expression is maintained in intracellular amastigotes, HASPB cannot be detected on the external surface in these cells. Thus HASPB may be a dual function protein that is shed by the infective metacyclic but retained internally once Leishmania are taken up by macrophages.     

Phenotypic changes associated with deletion and overexpression of a stage-regulated gene family in Leishmania

The LmcDNA16 locus of Leishmania major contains three highly related genes HASPA1 , HASPA2 and HASPB , encoding hydrophilic, acylated surface proteins and a tandem pair of unrelated sequences, SHERP1 and SHERP2 , coding for a small, hydrophilic protein that localizes to the endoplasmic reticulum and outer mitochondrial membrane. Differential regulation of these genes results in expression of a subset of the HASP proteins and SHERP only in infective stage parasites. To assess the contribution of these molecules to parasite virulence, the diploid LmcDNA16 gene locus has been removed by targeted gene deletion. Homozygous null mutants have precise deletions of both alleles and exhibit no HASP or SHERP expression. They are at least as virulent as wild-type parasites in macrophage invasion and intracellular survival assays, both in vitro and in vivo . Conversely, null mutants engineered to overexpress the entire LmcDNA16 gene locus are unable to survive within the intramacrophage environment despite their differentiation into infective metacyclic parasites. Both null and overexpressing null parasites show increased sensitivity to complement-mediated lysis, suggesting perturbation of their surface architecture. Avirulence in overexpressing parasites correlates with selective depletion of a specific lipid species, decreased expression of the major surface glycoprotein GP63, but no significant downregulation of the glycoconjugate lipophosphoglycan.     

A lipophosphoglycan-independent method for isolation of infective Leishmania metacyclic promastigotes by density gradient centrifugation.

At the end of their growth in the sand fly, Leishmania parasites differentiate into the infective metacyclic promastigote stage, which is transmitted to the mammalian host. Thus, in experimental studies of parasite infectivity toward animals or macrophages, the use of purified metacyclics is generally preferred. While metacyclics of several Leishmania species can be efficiently purified with the aid of lectins or monoclonal antibodies, which differentially exploit stage-specific differences in the structure of the abundant surface glycolipid lipophosphoglycan (LPG), such reagents are unavailable for most species and they are unsuitable for studies involving LPG-deficient mutants. Here we describe a simple density gradient centrifugation method, which allows the rapid purification of infective metacyclic parasites from both wild-type and LPG-deficient Leishmania major. The purified metacyclic promastigotes are authentic, as judged by criteria such as their morphology, expression of the metacyclic-specific gene SHERP, and ability to invade and replicate within macrophages in vitro. Preliminary studies suggest that this method is applicable to other Leishmania species including L. donovani.     

The stage‐regulated HASPB and SHERP proteins are essential for differentiation of the protozoan parasite Leishmania major in its sand fly vector,Phlebotomus papatasi

The stage‐regulated HASPB and SHERP proteins of Leishmania major are predominantly expressed in cultured metacyclic parasites that are competent for macrophage uptake and survival. The role of these proteins in parasite development in the sand fly vector has not been explored, however. Here, we confirm that expression of HASPB is detected only in vector metacyclic stages, correlating with the expression of metacyclic‐specific lipophosphoglycan and providing the first definitive protein marker for this infective sand fly stage. Similarly, SHERP is expressed in vector metacyclics but is also detected at low levels in the preceding short promastigote stage. Using genetically modified parasites lacking or complemented for the LmcDNA16 locus on chromosome 23 that contains the HASP and SHERP genes, we further show that the presence of this locus is essential for parasite differentiation to the metacyclic stage in Phlebotomus papatasi. While wild‐type and complemented parasites transform normally in late‐stage infections, generating metacyclic promastigotes and colonizing the sand fly stomodeal valve, null parasites accumulate at the earlier elongated nectomonad stage of development within the abdominal and thoracic midgut of the sand fly. Complementation with HASPB or SHERP alone suggests that HASPB is the dominant effector molecule in this process.     

Acylation-dependent protein export in Leishmania

The surface of the protozoan parasite Leishmania is unusual in that it consists predominantly of glycosylphosphatidylinositol-anchored glycoconjugates and proteins. Additionally, a family of hydrophilic acylated surface proteins (HASPs) has been localized to the extracellular face of the plasma membrane in infective parasite stages. These surface polypeptides lack a recognizable endoplasmic reticulum secretory signal sequence, transmembrane spanning domain, or glycosylphosphatidylinositol-anchor consensus sequence, indicating that novel mechanisms are involved in their transport and localization. Here, we show that the N-terminal domain of HASPB contains primary structural information that directs both N-myristoylation and palmitoylation and is essential for correct localization of the protein to the plasma membrane. Furthermore, the N-terminal 18 amino acids of HASPB, encoding the dual acylation site, are sufficient to target the heterologous Aequorea victoria green fluorescent protein to the cell surface of Leishmania. Mutagenesis of the predicted acylated residues confirms that modification by both myristate and palmitate is required for correct trafficking. These data suggest that HASPB is a representative of a novel class of proteins whose translocation onto the surface of eukaryotic cells is dependent upon a "non-classical" pathway involving N-myristoylation/palmitoylation. Significantly, HASPB is also translocated on to the extracellular face of the plasma membrane of transfected mammalian cells, indicating that the export signal for HASPB is recognized by a higher eukaryotic export mechanism.     

Endoplasmic reticulum stress-induced apoptosis in Leishmania through Ca2+-dependent and caspase-independent mechanism

Numerous reports have shown that mitochondrial dysfunctions play a major role in apoptosis of Leishmania parasites, but the endoplasmic reticulum (ER) stress-induced apoptosis in Leishmania remains largely unknown. In this study, we investigate ER stress-induced apoptotic pathways in Leishmania major using tunicamycin as an ER stress inducer. ER stress activates the expression of ER-localized chaperone protein BIP/GRP78 (binding protein/identical to the 78-kDa glucose-regulated protein) with concomitant generation of intracellular reactive oxygen species. Upon exposure to ER stress, the elevation of cytosolic Ca(2+) level is observed due to release of Ca(2+) from internal stores. Increase in cytosolic Ca(2+) causes mitochondrial membrane potential depolarization and ATP loss as ablation of Ca(2+) by blocking voltage-gated cation channels with verapamil preserves mitochondrial membrane potential and cellular ATP content. Furthermore, ER stress-induced reactive oxygen species (ROS)-dependent release of cytochrome c and endonuclease G from mitochondria to cytosol and subsequent translocation of endonuclease G to nucleus are observed. Inhibition of caspase-like proteases with the caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone or metacaspase inhibitor antipain does not prevent nuclear DNA fragmentation and phosphatidylserine exposure. Conversely, significant protection in tunicamycin-induced DNA degradation and phosphatidylserine exposure was achieved by either pretreatment of antioxidants (N-acetyl-L-cysteine, GSH, and L-cysteine), chemical chaperone (4-phenylbutyric acid), or addition of Ca(2+) chelator (1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid-acetoxymethyl ester). Taken together, these data strongly demonstrate that ER stress-induced apoptosis in L. major is dependent on ROS and Ca(2+)-induced mitochondrial toxicity but independent of caspase-like proteases.     

Membrane topology of the hepatitis C virus NS2 protein

The hepatitis C virus (HCV) NS2 protein is a hydrophobic protein. Previous studies indicate that this protein is an integral membrane protein, which is targeted to the membrane of the endoplasmic reticulum (ER) by the signal sequence located in its preceding p7 protein. In this report, we demonstrate that the membrane association of NS2 is p7-independent and occurs co-translationally. Further deletion-mapping studies suggest the presence of two internal signal sequences in NS2. These two internal signal sequences, which are located within amino acids 839-883 and amino acids 928-960, could target the alpha-globin reporter, a cytosolic protein, to the membrane compartments in HuH7 hepatoma cells. The presence of multiple signal sequences for its membrane association suggests that NS2 has multiple transmembrane domains. The glycosylation studies indicate that both amino and carboxyl termini of NS2 are located in the endoplasmic reticulum lumen. Based on these results, a model for the NS2 membrane topology is presented.     

The immunologically protective P-4 antigen of Leishmania amastigotes. A developmentally regulated single strand-specific nuclease associated with the endoplasmic reticulum

The purified membrane-associated Leishmania pifanoi amastigote protein P-4 has been shown to induce protective immunity against infection and to elicit preferentially a T helper 1-like response in peripheral blood mononuclear cells of patients with American cutaneous leishmaniasis. As this molecule is potentially important for future vaccine studies, the L. pifanoi gene encoding the P-4 membrane protein was cloned and sequenced. Southern blot analyses indicate the presence of six tandemly arrayed copies of the P-4 gene in L. pifanoi; homologues of the P-4 gene are found in all other species of the genus Leishmania examined. DNA-derived protein sequence data indicated an identity to the P1 zinc-dependent nuclease of Penicillium citrinum (20.8%) and the C-terminal domain of the 3' nucleotidase of Leishmania donovani (33.7%). Consistent with these sequence analyses, purified L. pifanoi P-4 protein possesses single strand nuclease (DNA and RNA) and phosphomonoesterase activity, with a preference for UMP > TMP > AMP > CMP. Double-labeling immunofluorescence microscopic analyses employing anti-binding protein antibodies revealed that the P-4 protein is localized in the endoplasmic reticulum of the amastigote. Northern blot analyses indicated that the gene is selectively expressed in the intracellular amastigote stage (mammalian host) but not in the promastigote stage (insect) of the parasite. Based upon its subcellular localization and single-stranded specific nuclease activity, possible roles of the P-4 nuclease in the amastigote in RNA stability (gene expression) or DNA repair are discussed.     

Unconventional secretory routes: direct protein export across the plasma membrane of mammalian cells

The vast majority of extracellular proteins are exported from mammalian cells by the endoplasmic reticulum/Golgi-dependent secretory pathway. For poorly understood reasons, however, a heterogenous group of extracellular proteins has been discovered that does not make use of signal peptide-dependent secretory transport. Both the release mechanisms and the molecular identity of the secretory machines involved have remained elusive. Recent studies now have established a subgroup of unconventional secretory proteins capable of translocating from the cytoplasm directly across the plasma membrane to get access to the exterior of eukaryotic cells. This review aims to focus on a detailed comparison of the subcellular site of membrane translocation of various unconventional secretory proteins such as the proangiogenic molecule fibroblast growth factor-2 (FGF-2) and Leishmania hydrophilic acylated surface protein B (HASP B). A potential link between membrane translocation and quality control as an integral part of unconventional secretory processes is discussed.     

Hemoglobin receptor in Leishmania is a hexokinase located in the flagellar pocket

Hb endocytosis in Leishmania is mediated through a 46-kDa protein located in the flagellar pocket. To understand the nature of the Hb receptor (HbR), we have purified the 46-kDa protein to homogeneity from Leishmania promastigote membrane. Purified HbR specifically binds Hb. The gene for HbR was cloned, and sequence analysis of the full-length HbR gene indicates the presence of hexokinase (HK) signature sequences, ATP-binding domain, and PTS-II motif. Four lines of evidence indicate that HbR in Leishmania is a hexokinase: 1) the recombinant HbR binds Hb, and the Hb-binding domain resides in the N terminus of the protein; 2) recombinant proteins and cell lysate prepared from HbR-overexpressing Leishmania promastigotes show enhanced HK activity in comparison with untransfected cells; 3) immunolocalization studies using antibodies against the N-terminal fragment (Ld-HbR-DeltaC) of Ld-HbR indicate that this protein is located in the flagellar pocket of Leishmania; and 4) binding and uptake of (125)I-Hb by Leishmania is significantly inhibited by anti-Ld-HbR-DeltaC antibody and Ld-HbR-DeltaC, respectively. Taken together, these results indicate that HK present in the flagellar pocket of Leishmania is involved in Hb endocytosis.     

Leishmania major elongation factor 1B complex has trypanothione S-transferase and peroxidase activity

In the Trypanosomatidae, trypanothione has subsumed many of the roles of glutathione in defense against chemical and oxidant stress. Crithidia fasciculata lacks glutathione S-transferase, but contains an unusual trypanothione S-transferase activity that is associated with eukaryotic translation elongation factor 1B (eEF1B). Here we describe the cloning, expression, and reconstitution of the purified alpha, beta, and gamma subunits of eEF1B from Leishmania major. Individual subunits lacked trypanothione S-transferase activity. Only eEF1B, formed by reconstitution or co-expression of the three subunits, was able to conjugate a variety of electrophilic substrates to trypanothione or glutathionylspermidine, but not glutathione. In contrast to the C. fasciculata eEF1B, the L. major enzyme also displayed peroxidase activity against a variety of organic hydroperoxides. The enzyme showed no activity with hydrogen peroxide and greatest activity with linoleic acid hydroperoxide (1 unit mg(-1)). Kinetic studies suggest a ternary complex mechanism, with Km values of 140 mum for trypanothione and 7.4 mm for cumene hydroperoxide and kcat=25 s(-1). Immunofluorescence studies indicate that the enzyme may be localized to the surface of the endoplasmic reticulum. These results suggest that, in addition to its role in protein synthesis, the Leishmania eEF1B may help protect the parasite from lipid peroxidation.     

SRP-mediated protein targeting: structure and function revisited

The signal recognition particle (SRP) and its membrane-bound receptor (SR) deliver membrane proteins and secretory proteins to the translocation channel in the plasma membrane (or the endoplasmic reticulum). The general outline of the SRP pathway is conserved in all three kingdoms of life. During the past decade, structure determination together with functional studies has brought our understanding of the SRP-mediated protein transport to an almost molecular level. An impressive amount of new information especially on the prokaryotic SRP is integrated into the current picture of the SRP pathway.     

Direct Formation of Vaccinia Virus Membranes from the Endoplasmic Reticulum in the Absence of the Newly Characterized L2-Interacting Protein A30.5

Crescents consisting of a single lipoprotein membrane with an external protein scaffold comprise the initial structural elements of poxvirus morphogenesis. Crescents enlarge to form spherical immature virions, which enclose viroplasm consisting of proteins destined to form the cores of mature virions. Previous studies suggest that the L2 protein participates in the recruitment of endoplasmic reticulum (ER)-derived membranes to form immature virions within assembly sites of cytoplasmic factories. Here we show that L2 interacts with the previously uncharacterized 42-amino-acid A30.5 protein. An open reading frame similar in size to the one encoding A30.5 is at the same genome location in representatives of all chordopoxvirus genera. A30.5 has a putative transmembrane domain and colocalized with markers of the endoplasmic reticulum and with L2. By constructing a complementing cell line expressing A30.5, we isolated a deletion mutant virus that exhibits a defect in morphogenesis in normal cells. Large electron-dense cytoplasmic inclusions and clusters of scaffold protein-coated membranes that resemble crescents and immature virions devoid of viroplasm were seen in place of normal structures. Crescent-shaped membranes were continuous with the endoplasmic reticulum membrane and oriented with the convex scaffold protein-coated side facing the lumen, while clusters of completed spherical immature-virion-like forms were trapped within the expanded lumen. Immature-virion-like structures were more abundant in infected RK-13 cells than in BS-C-1 or HeLa cells, in which cytoplasmic inclusions were decorated with scaffold protein-coated membrane arcs. We suggest that the outer surface of the poxvirus virion is derived from the luminal side of the ER membrane.     

A plasmodesmata-associated beta-1,3-glucanase in Arabidopsis

Plasmodesmal conductivity is regulated in part by callose turnover, which is hypothesized to be determined by beta-1,3-glucan synthase versus glucanase activities. A proteomic analysis of an Arabidopsis thaliana plasmodesmata (Pd)-rich fraction identified a beta-1,3-glucanase as present in this fraction. The protein encoded by the putative plasmodesmal associated protein (ppap) gene, termed AtBG_ppap, had previously been found to be a post-translationally modified glycosylphosphatidylinositol (GPI) lipid-anchored protein. When fused to green fluorescent protein (GFP) and expressed in tobacco (Nicotiana tabacum) or Nicotiana benthamiana epidermal cells, this protein displays fluorescence patterns in the endoplasmic reticulum (ER) membrane system, along the cell periphery and in a punctate pattern that co-localizes with aniline blue-stained callose present around the Pd. Plasma membrane localization was verified by co-localization of AtBG_ppap:GFP together with a plasma membrane marker N-[3-triethylammoniumpropyl]-4-[p-diethylaminophenylhexatrienyl] pyridinium dibromide (FM4-64) in plasmolysed cells. In Arabidopsis T-DNA insertion mutants that do not transcribe AtBG_ppap, functional studies showed that GFP cell-to-cell movement between epidermal cells is reduced, and the conductivity coefficient of Pd is lower. Measurements of callose levels around Pd after wounding revealed that callose accumulation in the mutant plants was higher. Taken together, we suggest that AtBG_ppap is a Pd-associated membrane protein involved in plasmodesmal callose degradation, and functions in the gating of Pd.     

Effects of altered cytoplasmic domains on transport of the vesicular stomatitis virus glycoprotein are transferable to other proteins.

Alterations of the cytoplasmic domain of the vesicular stomatitis virus glycoprotein (G protein) were shown previously to affect transport of the protein from the endoplasmic reticulum, and recent studies have shown that this occurs without detectable effects on G protein folding and trimerization (R. W. Doms et al., J. Cell Biol., in press). Deletions within this domain slowed exit of the mutant proteins from the endoplasmic reticulum, and replacement of this domain with a foreign 12-amino-acid sequence blocked all transport out of the endoplasmic reticulum. To extend these studies, we determined whether such effects of cytoplasmic domain changes were transferable to other proteins. Three different assays showed that the effects of the mutations on transport of two membrane-anchored secretory proteins were the same as those observed with vesicular stomatitis virus G protein. In addition, possible effects on oligomerization were examined for both transported and nontransported forms of membrane-anchored human chorionic gonadotropin-alpha. These membrane-anchored forms, like the nonanchored human chorionic gonadotropin-alpha, had sedimentation coefficients consistent with a monomeric structure. Taken together, our results provide strong evidence that these cytoplasmic mutations affect transport by affecting interactions at or near the cytoplasmic side of the membrane.     

Role of the nuclear envelope in synthesis, processing, and transport of membrane glycoproteins

The outer nuclear membrane is morphologically similar to rough endoplasmic reticulum. The presence of ribosomes bound to its cytoplasmic surface suggests that it could be a site of synthesis of membrane glycoproteins. We have examined the biogenesis of the vesicular stomatitis virus G protein in the nuclear envelope as a model for the biogenesis of membrane glycoproteins. G protein was present in nuclear membranes of infected Friend erythroleukemia cells immediately following synthesis and was transported out of nuclear membranes to cytoplasmic membranes with a time course similar to transport from rough endoplasmic reticulum (t 1/2 = 5-7 min). Temperature-sensitive mutations in viral membrane proteins which block transport of G protein from endoplasmic reticulum also blocked transport of G protein from the nuclear envelope. Friend erythroleukemia cells and NIH 3T3 cells differed in the fraction of newly synthesized G protein found in nuclear membranes, apparently reflecting the relative amount of nuclear membrane compared to endoplasmic reticulum available for glycoprotein synthesis. Nuclear membranes from erythroleukemia cells appeared to have the enzymatic activities necessary for cleavage of the signal sequence and core glycosylation of newly synthesized G protein. Signal peptidase activity was detected by the ability of detergent-solubilized membranes of isolated nuclei to correctly remove the signal sequence of human preplacental lactogen. RNA isolated from the nuclear envelope was highly enriched for G protein mRNA, suggesting that G protein was synthesized on the outer nuclear membrane rather than redistributing to nuclear membranes from endoplasmic reticulum before or during cell fractionation. These results suggest a mechanism for incorporation of membrane glycoproteins into the nuclear envelope and suggest that in some cell types the nuclear envelope is a major source of newly synthesized membrane glycoproteins.     

Biophysical properties of lipids and dynamic membranes

The lipid bilayer is a 3D assembly with a rich variety of physical features that modulate cell signaling and protein function. Lateral and transverse forces within the membrane are significant and change rapidly as the membrane is bent or stretched and as new constituents are added, removed or chemically modified. Recent studies have revealed how differences in structure between the two leaflets of the bilayer and between different areas of the bilayer can interact together with membrane deformation to alter the activities of transmembrane channels and peripheral membrane binding proteins. Here, we highlight some recent reports that the physical properties of the membrane can help control the function of transmembrane proteins and the motor-dependent elongation of internal organelles, such as the endoplasmic reticulum.     

Acyl-CoA synthetase isoforms 1, 4, and 5 are present in different subcellular membranes in rat liver and can be inhibited independently

Inhibition studies have suggested that acyl-CoA synthetase (ACS, EC ) isoforms might regulate the use of acyl-CoAs by different metabolic pathways. In order to determine whether the subcellular locations differed for each of the three ACSs present in liver and whether these isoforms were regulated independently, non-cross-reacting peptide antibodies were raised against ACS1, ACS4, and ACS5. ACS1 was identified in endoplasmic reticulum, mitochondria-associated membrane (MAM), and cytosol, but not in mitochondria. ACS4 was present primarily in MAM, and the 76-kDa ACS5 protein was located in mitochondrial membrane. Consistent with these locations, N-ethylmaleimide, an inhibitor of ACS4, inhibited ACS activity 47% in MAM and 28% in endoplasmic reticulum. Troglitazone, a second ACS4 inhibitor, inhibited ACS activity <10% in microsomes and mitochondria and 45% in MAM. Triacsin C, a competitive inhibitor of both ACS1 and ACS4, inhibited ACS activity similarly in endoplasmic reticulum, MAM, and mitochondria, suggesting that a hitherto unidentified triacsin-sensitive ACS is present in mitochondria. ACS1, ACS4, and ACS5 were regulated independently by fasting and re-feeding. Fasting rats for 48 h resulted in a decrease in ACS4 protein, and an increase in ACS5. Re-feeding normal chow or a high sucrose diet for 24 h after a 48-h fast increased both ACS1 and ACS4 protein expression 1.5-2.0-fold, consistent with inhibition studies. These results suggest that ACS1 and ACS4 may be linked to triacylglycerol synthesis. Taken together, the data suggest that acyl-CoAs may be functionally channeled to specific metabolic pathways through different ACS isoforms in unique subcellular locations.     

DSCR2, a Down syndrome critical region protein, is localized to the endoplasmic reticulum of mammalian cells

We used immunocytochemical and fluorescence assays to investigate the subcellular location of the protein encoded by Down syndrome critical region gene 2 (DSCR2) in transfected cells. It was previously suggested that DSCR2 is located in the plasma membrane as an integral membrane protein. Interestingly, we observed this protein in the endoplasmic reticulum (ER) of cells. We also studied whether the truncated forms of DSCR2 showed different subcellular distributions. Our observations indicate that DSCR2 probably is not inserted into the membrane of the endoplasmic reticulum since the fragments lacking the predicted transmembrane (TM) helices remained associated with the ER. Our analyses suggest that, although DSCR2 is associated with the endoplasmic reticulum, it is not an integral membrane protein and it is maintained on the cytoplasmic side of the ER by indirect interaction with the ER membrane or with another protein.