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Eukaryotic Cell, October 2007, p. 1865-1875, Vol. 6, No. 10
1535-9778/07/$08.00+0     doi:10.1128/EC.00134-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Down-Regulation of the Trypanosomatid Signal Recognition Particle Affects the Biogenesis of Polytopic Membrane Proteins but Not of Signal Peptide-Containing Proteins{triangledown}

Yaniv Lustig,1,{dagger} Yaron Vagima,1,{dagger} Hanoch Goldshmidt,1,{dagger} Avigail Erlanger,1 Vered Ozeri,1 James Vince,2 Malcolm J. McConville,2 Dennis M. Dwyer,3 Scott M. Landfear,4 and Shulamit Michaeli1*

The Mina and Everard Goodman Faculty of Life Science, Bar Ilan University, Ramat-Gan 52900, Israel,1 Department of Biochemistry and Molecular Biology, University of Melbourne, Bio21 Molecular Sciences and Biotechnology Institute, Parkville, Victoria, Australia,2 Cell Biology Section, Laboratory of Parasitic Diseases, NIAID, National Institutes of Health, Bethesda, Maryland,3 Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, Oregon 972394

Received 23 April 2007/ Accepted 23 July 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein translocation across the endoplasmic reticulum is mediated by the signal recognition particle (SRP). In this study, the SRP pathway in trypanosomatids was down-regulated by two approaches: RNA interference (RNAi) silencing of genes encoding SRP proteins in Trypanosoma brucei and overexpression of dominant-negative mutants of 7SL RNA in Leptomonas collosoma. The biogenesis of both signal peptide-containing proteins and polytopic membrane proteins was examined using endogenous and green fluorescent protein-fused proteins. RNAi silencing of SRP54 or SRP68 in T. brucei resulted in reduced levels of polytopic membrane proteins, but no effect on the level of signal peptide-containing proteins was observed. When SRP deficiency was achieved in L. collosoma by overexpression of dominant-negative mutated 7SL RNA, a major effect was observed on polytopic membrane proteins but not on signal peptide-containing proteins. This study included two trypanosomatid species, tested various protein substrates, and induced depletion of the SRP pathway by affecting either the levels of SRP binding proteins or that of SRP RNA. Our results demonstrate that, as in bacteria but in contrast to mammalian cells, the trypanosome SRP is mostly essential for the biogenesis of membrane proteins.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The signal recognition particle (SRP) is a key component in the biogenesis of membrane and signal peptide (SP)-containing proteins across the endoplasmic reticulum (ER) in eukaryotes and through the bacterial plasma membrane (18). SRP binds to the hydrophobic SP or transmembrane (TM) domain emerging from the ribosome, which results in a slowing down or a pause in translation, a phenomenon termed "elongation arrest" (9, 32, 38, 42). The SRP then targets the nascent-chain ribosome complex to the endoplasmic reticulum (ER) membrane by interacting with the membrane-bound SRP receptor (11). After this GTP-dependent docking event, translocation of the polypeptide takes place cotranslationally through the translocon (27, 28, 40). The well-defined mammalian SRP is composed of a single RNA molecule, the 7SL RNA, and six proteins (39). In bacteria such as Escherichia coli, the SRP is composed of a single RNA molecule (4.5S RNA) and a single protein, the fifty-four homologue (Ffh) (33).

The in vivo role of SRP in protein translocation was initially elucidated for bacteria and yeast, but recently it was also studied with cultured mammalian cells. Disruption of the SRP pathway in E. coli is lethal, mostly because of defects in the translocation of inner membrane proteins (35, 37). The secretion of SP-containing proteins such as periplasmic or outer membrane proteins is only slightly affected, and most of these proteins are translocated by the posttranslational pathway (20). In the yeast Saccharomyces cerevisiae, SRP is not essential for cell growth (5, 30). Cells lacking SRP grow poorly and are severely impaired in the translocation of proteins across the ER. However, different proteins show translocation defects of varying severity (5, 13, 36). It has been suggested that in yeast, the choice of the targeting pathway is dependent on the hydrophobicity of the signal sequence (20, 31). In higher eukaryotes, targeting of both secreted proteins and membrane proteins is thought to be SRP dependent (17). Depletion of the SRP pathway by RNA interference (RNAi) knockdown of SRP proteins in cultured mammalian cells indicated that two death receptors of the TRAIL family, DR4 and DR5, are differentially affected. This study suggested that not only does the SRP mediate the translocation of proteins across the ER, but an active posttranslational pathway also exists (34). More recently, it was demonstrated that RNAi silencing of SRP proteins in cultured mammalian cells results in accumulation of several proteins in the secretory pathway, indicating post-ER trafficking defects. Major defects were observed in anterograde transport, retrograde transport, and recycling from the endosome to the plasma membrane. Reductions in the levels of both SP-containing proteins and membrane proteins were observed, albeit to different degrees (19).

Relatively little is known about the SRP pathway in trypanosomes. The trypanosome SRP, as opposed to all other SRPs in nature, is composed of two RNA molecules, the 7SL RNA and a special tRNA-like molecule, sRNA-76 in Trypanosoma brucei and sRNA-85 in Leptomonas collosoma (1, 23). Recent purification of the T. brucei SRP suggests that the RNP is composed of only the four S-domain proteins SRP19, SRP68/72, and SRP54 and lacks the Alu domain binding proteins present in other eukaryotes (25). We proposed that the tRNA-like molecule may functionally substitute for the lack of a functional Alu domain in the trypanosome SRP (25). Another unusual feature is that the L. collosoma 7SL RNA is found in the cell in two stable conformations: one that is enriched in the ribosome fraction (7SL I), and the other in the free SRP (7SL II) (2, 3). The conversion of 7SL II to 7SL I is associated with a novel RNA-editing event of C-to-U conversion in domain III of the RNA (3). RNAi silencing to deplete SRP proteins in T. brucei suggests that the SRP pathway is essential for growth. Surprisingly, despite the severe depletion of SRP, SP-containing proteins such as the surface EP, the lysosomal protein p67, and the flagellar pocket protein CRAM traverse the ER membrane but are mislocalized and accumulate in megavesicles, probably because of a secondary effect on protein sorting. In the silenced cells, the proteins are correctly glycosylated, suggesting that these proteins traverse the ER membrane and that their mislocalization may stem from secondary defects in the biogenesis of membrane proteins. Defects in the processing of membrane proteins could change the composition both of intracellular membranes such as the ER and Golgi apparatus and of the plasma membrane and may affect intracellular trafficking. Such defects may affect the sorting of the proteins and hence cause their mislocalization as a secondary defect elicited by SRP depletion. Although most translocated proteins are mislocalized following SRP depletion, their cellular levels at steady state are unchanged. The translocation of these proteins to the ER under conditions of SRP depletion suggests that an active alternative pathway for protein translocation must exist (24).

In this study, the SRP pathway was perturbed in T. brucei by RNAi silencing of SRP proteins and in L. collosoma by expression of dominant-negative mutants of 7SL RNA. Our data suggest that although depletion of SRP was achieved in different ways, the expression and localization of multispanning TM domain proteins were severely impaired in both types of SRP-deficient cells. However, the translocation of proteins carrying an SP was not perturbed, suggesting that, as in E. coli (35, 37) but unlike the situation in mammalian cells (19), the trypanosome SRP is essential mainly for the biogenesis of membrane proteins.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Oligonucleotides used in this study. The following primers were used: 35171 (5'-ACG TCA ACG CCG CAA CGG AAT TCC GGC ACC CTA TGC TGG TCC AG-3'; antisense, carrying an EcoRI site for generating the A1 mutation), 35173 (5'-CTT TAA CGG AGG GGG CCG GAA TTC CGG CGC CCC TTC TGC AAG GG-3'; antisense, carrying an EcoRI site for generating the A3 mutation), 35174 (5'-GAC CCA GTT CCT CTA CCG GAA TTC CGT TTA ACG GAG GGG GCG CG-3'; antisense, carrying an EcoRI site for generating the A7 mutation), 15366 (5'-GCT CTA GAG TCG ACT TTG ACC ACC CAT TAT C-3'; sense, carrying an XbaI site for generating the mega primer), and 15137 (5'-CCG GAT CCG GTG CGA TGA AAT GAG ACG G-3'; antisense, carrying a BamHI site for PCR amplifying the mega primer [inserting the mutation]). The A2 and A4 mutations were generated as previously described (2). For TbNT8.1-GFP, primers were blastAscI (5'-GAC TGG CGC CAT GGC CAA GCC TTT GTC TCA AAG-3'; sense, including an AscI site) and BlastPacI (5'-GAC GTT AAT TAA ACG GTT AAT TTA GCA CGT GTC AG-3'; antisense, including a PacI site). Other primers used were 18288 (5'-TGC GGG CGT CCT ACC-3'; antisense, complementary to positions of sRNA-85), 5303 (5'-GCA GAGCAC CAC GTC AAC CGC-3'; antisense, complementary to positions of 7SL RNA), GT3-GFP 5 (5'-GACTG GATCCATGAGCGACAAGTTGGAGGCGAACGTGCAG-3'; sense, including a BamHI site), and GT3-GFP 6 (5'-ATCCATTTCTTTC TTCCCGACGAATTC-3'; antisense, including an EcoRV site).

Generation of constructs, transformation, and generation of transgenic parasites. The construction of the A2 and A4 mutants has been described previously (2). Mutants A3 to A7 were constructed by PCR mutagenesis using primers carrying the antisense mutations and a 5' primer situated upstream of the tRNAArg extragenic promoter of the 7SL RNA gene. The resulting PCR product carrying the mutation (mega primer) was used to amplify the remaining gene using the antisense primer. The PCR product was cloned into the BamHI site of the pX vector.

The SP-GFP-TM plasmid was generated as described elsewhere (10). To generate pX-GT3-GFP, the gene was amplified using the forward and reverse primers (see the list above) and the fragment was cloned between the BamHI and EcoRV sites of pXG'-GFP. L. collosoma cells were transfected with 50 to 100 µg of plasmid DNA, and stable cell lines were generated, as described previously (12). To generate cell lines expressing both 7SL RNA mutants and green fluorescent protein (GFP)-fused proteins, cells were first transfected with the pX-hygro plasmid carrying the 7SL RNA mutant. Clones selected on 1 mg/ml hygromycin were further transfected with the pX-neo plasmid carrying the GFP-fused proteins and selected on 500 µg/ml G418. The generation of T. brucei transgenic parasites expressing a construct to silence SRP54 and SRP68 has been described previously (24, 25). To create a cell line expressing both the silencing construct for SRP54 and the nucleobase transporter, the GFP-TbNT8.1 construct cloned into pXS2 (described in reference 14) was modified by replacing the neomycin resistance gene with a blasticidin resistance gene, amplifying the blasticidin gene using the primers specified in the list above, and cloning it between the AscI and PacI sites in the pXS2 plasmid.

Isolation of membrane-bound and soluble proteins. (i) L. collosoma. Cells (2 x 108) were harvested, washed with ice-cold phosphate-buffered saline (PBS), and resuspended in 200 µl of Triton X-100 lysis buffer (1% Triton X-100, 25 mM HEPES [pH 7.4], 1 mM EDTA) containing a protease inhibitor cocktail (Roche) at 4°C for 30 min. The extract was centrifuged (at 14,000 x g for 20 min at 4°C), and the pellet was washed with ice-cold PBS. Proteins in the detergent (membrane fraction) and aqueous (soluble fraction) phases were precipitated with 9 volumes of acetone (–20°C, 16 h).

(ii) T. brucei. Cells (5 x 107) were harvested, washed twice with ice-cold PBS, and resuspended in 1 ml hypotonic buffer (1 mM HEPES-KOH [pH 7.5], 1 mM EDTA) containing a protease inhibitor cocktail (Roche) at 4°C for 10 min. Next, 200 µl of lysis buffer (25 mM HEPES KOH [pH 7.5], 1 mM EDTA, 100 mM sucrose, 80 mM potassium acetate) was added, and the extract was passed through a 27-gauge syringe three to five times. The lysate was centrifuged (at 1,000 x g for 10 min at 4°C), and the pellet (membrane fraction) and supernatant (soluble fraction) were separated and acetone precipitated.

Protein extraction of polytopic membrane proteins. To ensure the release of polytopic membrane proteins from the plasma membrane and the solubilization of aggregated membrane proteins, cells (8 x 107) were harvested, washed twice with ice-cold PBS, and resuspended in lysis buffer (10 mM HEPES-KOH [pH 7.4], 300 mM KCl, 1% Triton X-100, 1 mM EGTA, 0.1% sodium dodecyl sulfate [SDS]) containing a protease inhibitor cocktail (Roche) at 4°C for 10 min.

In vivo labeling and immunoprecipitation. Cells were grown at 27°C in semidefined medium-79 (SDM-79). The cells were washed twice with PBS and resuspended at 108/ml in prewarmed (27°C) Met-Cys-depleted Dulbecco's modified Eagle's medium (Invitrogen). The cells were labeled with 200µCi/ml L-[35S]Met-Cys (Hybond; Amersham Biosciences) for 3 h. Labeled cells were washed once with PBS and solubilized in 1 ml of lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS) containing a protease inhibitor mixture (Sigma). The extract was precleared by centrifugation. Antibodies against the vacuolar-type proton-translocating pyrophosphatase (VH+ppase) (5 µl/107 cells) were bound to protein A-Sepharose beads (Santa Cruz Biotechnology) by rotating for 1 h at room temperature and were washed once with lysis buffer. Finally, 50 µl of an antibody-protein A-Sepharose suspension was mixed with 0.5 ml of cell lysate (from 109 cells) at 4°C for 12 h. After the supernatant was removed, the pellet was washed four times with lysis buffer and once with TEN buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 5 mM EDTA). The bound proteins were separated on a 10% SDS-polyacrylamide gel.

Northern blot analysis. Total RNA (10 to 20 µg) was fractionated on a 10% polyacrylamide-7 M urea denaturing gel and electroblotted onto a nylon membrane (Hybond; Amersham Biosciences). Hybridization with a {gamma}-32P-end-labeled oligonucleotide was performed at 37°C in 2x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% SDS-5x Denhardt's solution-100 µg/ml salmon sperm DNA.

Western blot analysis. Whole-cell extracts or membrane and soluble fractions were separated on an SDS-polyacrylamide gel, transferred to a PROTAN membrane (Whatman Schleicher & Schuell), and probed with either anti-GFP (diluted 1:3,000), anti-BiP (diluted 1:500), anti-hnRNPD0 (diluted 1:5,000), anti-EP (diluted 1:2,000), anti-Tb29 (diluted 1:1,000), or anti-VH+ppase (diluted 1: 2,500) antibodies. The bound antibodies were detected with goat anti-rabbit immunoglobulin G (IgG) or anti-mouse IgG coupled to horseradish peroxidase and were visualized by ECL (Amersham Biosciences).

Confocal microscopy. Live-cell fluorescence microscopy was performed by labeling the endocytic compartments with the vital dye FM-4-64. FM-4-64 (final concentration, 10 µM) was mixed with parasites, and the cells were examined after 40 min. Cells were immobilized in 1.5 to 2% low-melting-point agarose (Sigma) on poly-L-lysine-coated coverslips and were visualized under a Zeiss LSM 510 META inverted microscope.

For immunofluorescence, cells were washed with PBS, mounted on poly-L-lysine-coated slides, and fixed with 4% formaldehyde in PBS at room temperature for 30 min. Cells were first incubated with PBS containing 10% fetal calf serum at room temperature for 30 min and then incubated with the primary antibodies (anti-VH+ppase [1:500] or anti-BiP [1:200]) in PBS-0.1% Nonidet P-40 for 1 h. After a wash with PBS, the cells were reacted with anti-IgG conjugated to fluorescein isothiocyanate (FITC; Jackson ImmunoResearch). To stain the nucleus and kinetoplast, the cells were incubated with 4',6'-diamidino-2-phenylindole (DAPI) for 5 min. The cells were visualized under a Zeiss LSM 510 META inverted microscope.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
RNAi of SRP proteins severely affects the biogenesis of polytopic membrane proteins. We previously described the consequences of depleting the SRP pathway, which is essential in trypanosomes (24, 25). In SRP54-silenced cells, SP-containing proteins traverse the ER membrane and their cellular levels are unchanged, but most of the proteins are mislocalized and do not reach their final destination. We proposed that the defects observed in intracellular trafficking originate from defects in polytopic membrane protein biogenesis (24, 25). To further examine this hypothesis, we studied the biogenesis of polytopic membrane proteins when either SRP54 or SRP68 was depleted. Silencing of either gene results in similar protein translocation defects (25). Three polytopic membrane proteins were examined: TbNT8.1 (14), Tb29-2 (21), and VH+ppase (15).

TbNT8.1 is a permease that transports hypoxanthine, adenine, guanine, and xanthine and is composed of 11 TM domains. This permease is expressed more abundantly in procyclic-form than in bloodstream-form parasites and thus is regulated during the parasite life cycle. The protein was fused to GFP at the N terminus, and the fusion protein was found to localize properly to the plasma membrane (14). Transgenic parasites carrying the SRP54-silencing construct (24) were transfected with the plasmid carrying the GFP-fused TbNT8.1 transporter expressed from the EP promoter and carrying the blasticidin resistance gene. The expression of TbNT8.1 was examined in uninduced and silenced cells (3 days after induction). The parasites were lysed, and membrane-bound and soluble proteins were examined by Western blot analysis with anti-GFP antibodies. The quality of the fractionation was demonstrated by the distribution of the cytoplasmic RNA binding protein hnRNPD0. The results in Fig. 1A1 show that while the GFP-TbNT8.1 fusion protein was detected at the expected size in uninduced cells, it was barely detected following SRP54 depletion. In addition, a polypeptide of ~28 kDa that reacted with anti-GFP antibodies was released via proteolysis from the fusion protein. This proteolytically resistant GFP product, also known as prGFP (8, 29), was found only in the soluble fraction, as expected, since only the fusion protein carrying TM domains is expected to reside in the membrane fraction. The level of EP was used to examine the effect of the depletion on SP-containing proteins. Our results indicate very minor changes in the level of this glycosylphosphatidylinositol-anchored protein upon SRP depletion, in agreement with previous results that detected minimal changes in the cellular level of EP following SRP54 or SRP68 depletion (24, 25). These data indicate major changes in the biogenesis of polytopic membrane proteins, but not in that of SP-containing proteins, in SRP-depleted cells. We further examined the localization of GFP-TbNT8.1 by confocal microscopy. The results, presented in Fig. 1A2, indicate that in uninduced cells, the transporter is found in the plasma membrane, but after 3 days of SRP depletion, the GFP-fused transporter is hardly detected.


Figure 1
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  		FIG. 1. Levels of polytopic membrane proteins under conditions of SRP knockdown. (A) Effect on TbNT8.1-GFP. (A1) Membrane and soluble fractions were prepared as described in Materials and Methods from uninduced (–Tet) and SRP54-silenced (+Tet) cells on the third day after induction and were subjected to Western blot analysis with anti-GFP and anti-EP antibodies. Anti-hnRNPD0 antibodies were used to control for the quality of fractionation. The positions of fused as well as cleaved GFP are indicated. (A2) Cells were fixed with 4% formaldehyde for 25 min and visualized by confocal microscopy. DIC, differential interference contrast; IFA, immunofluorescence assay. Scale bar, 5 µm. (B) Effect on VH+ppase. (B1) Western blot analysis of lysates from uninduced cells and SRP54-silenced cells on the third day after induction with anti-VH+ppase antibodies. hnRNPD0 was used to control for equal loading. (B2) Cells were fixed with 4% formaldehyde for 25 min, incubated with anti-VH+ppase antibodies, and detected by an FITC-conjugated secondary antibody. Cells were visualized by confocal microscopy. Scale bar, 5 µm. (C) Effect on Tb29 proteins. Western blot analysis of uninduced cells and SRP68-silenced cells was performed on the third day after induction with anti-Tb29 antibodies. Tb29-2 is the Tb29 protein containing the TM domain; Tb29-1 is the Tb29 protein carrying an SP. hnRNPD0 reactivity with antibodies was used to control for equal loading. (D) Effect of inhibition of proteolysis by the proteasome or lysosome on VH+ppase. Uninduced and SRP68-silenced cells on the third day after induction were treated with 5 µM lactacystin or 0.25 µM bafilomycin for 4 h. Proteins were prepared as described in Materials and Methods from lactacystin (a)- or bafilomycin (b)-treated uninduced or SRP68-silenced cells and were subjected to Western blot analysis with anti-VH+ppase antibodies. hnRNPD0 reactivity with antibodies was used to control for equal loading. (E) Immunoprecipitation of in vivo-labeled VH+ppase protein. Uninduced and induced cells on the third day after tetracycline induction were labeled with L-[35S]Met-Cys for 3 h. Extracts were prepared, and immunoprecipitation was performed, as described in Materials and Methods. The total-cell extract (Total) and the immunoprecipitated products (IP) were analyzed on a 10% SDS-polyacrylamide gel. The sizes of marker proteins are given on the left. The specific immunoprecipitated product is indicated by an arrow.

 
To further investigate the effect of SRP depletion on endogenous membrane protein biogenesis, the level and localization of the acidocalcisome VH+ppase were examined. The acidocalcisome is a vesicle rich in calcium, phosphorus, and magnesium. The VH+ppase functions in the acidification of these organelles (15). The protein possesses an N-terminal SP and 16 TM domains. It was previously suggested that this protein is translocated to the ER and is trafficked within the secretory pathway to the acidocalcisome (15). The level of VH+ppase was markedly reduced in SRP54-depleted cells, as observed by Western blot analysis using anti-VH+ppase (Fig. 1B1) as well as by immunofluorescence (Fig. 1B2).

We next examined the levels of two additional membrane proteins, Tb29-1 and Tb29-2. The Tb29 genes encode proteins that carry a large domain with an octapeptide repeat, similar to the S antigen of Plasmodium falciparum. Both proteins contain a unique N-terminal domain followed by the ERALRAEE octapeptide repeat. Tb29-2 contains, in addition, a large, unique C-terminal domain, with eight large hydrophobic TM domains flanked by positively charged amino acids. Tb29-1 does not contain any obvious TM domains. Antibodies generated against Tb29 recognize both Tb29-1 and Tb29-2. Localization studies indicated that the antibody stains proteins that are located below the flagellar pocket (22). The antibodies were used to probe a Western blot prepared from lysates of cells in which SRP68 had been silenced. The results (Fig. 1C) indicate that, as expected, the antibodies recognize two polypeptides, TB29-2 (270 kDa) and Tb29-1 (130 kDa). Upon silencing, only the expression of the protein containing the multiple TM domains was severely affected, suggesting that in SRP-depleted cells, only the levels of TM proteins are affected, not those of SP-containing proteins.

The inability to detect the polytopic membrane proteins (TbNT8.1, TB29-2, and VH+ppase) following silencing may suggest that misfolded proteins either are subjected to proteolysis or are aggregated and cannot enter the gel system. To investigate whether this degradation is mediated by the proteasome, uninduced cells and SRP68-silenced cells were treated with lactacystin (a proteasome inhibitor). The proteins were extracted from the cells with a buffer containing a high concentration of Triton X-100, which can possibly disrupt aggregates of proteins that were synthesized but failed to translocate the ER membrane. The level of VH+ppase was examined by Western blot analysis, and no difference in the level of the protein in the silenced cells upon treatment with lactacystin was observed (Fig. 1Da), suggesting that the degradation of the protein is not mediated by the proteasome (see Discussion).

We next examined if VH+ppase degradation is mediated by the lysosome. Unfolded proteins that failed to enter the ER may use the autophagy pathway (4) and travel from the face of the ER to the lysosome. Uninduced cells and SRP68-silenced cells were treated with bafilomycin A1, a specific inhibitor that inhibits acidification and protein degradation in lysosomes (43). The level of VH+ppase was examined by Western blot analysis. The results (Fig. 1Db) indicate no difference in the reduction of VH+ppase levels, suggesting that the degradation of the protein does not take place in the lysosome. To better understand why the protein was undetectable at steady state and to examine if the protein might be synthesized but then subjected to proteolysis, we examined the nascent synthesis of the VH+ppase under conditions of SRP silencing. Uninduced and silenced cells were labeled in vivo with L-[35S]Met-Cys as described in Materials and Methods. Extracts were prepared in the presence of detergents that are expected to disrupt aggregated proteins. The production of nascently synthesized VH+ppase was then examined by immunoprecipitation. The results (presented in Fig. 1E) indicate no difference in the amount of the nascently synthesized VH+ppase protein. Although the level of the immunoprecipitated protein from SRP-depleted cells was reduced by 40%, this reduction was due to the overall lower level of translation in the silenced cells, as can be seen by comparing the protein profile and the intensity of the incorporation obtained from the same number of cells. These results suggest that the failure to detect polytopic membrane proteins at steady state is not due to a specific and significant reduction in the level of their synthesis but that these proteins, which fail to integrate into the ER membrane, are most probably misfolded. These misfolded proteins either are subjected to degradation or form aggregates that cannot be disrupted under the detergent conditions used (see Discussion).

SRP depletion in L. collosoma by overexpressing mutant 7SL RNA, creating a dominant-negative effect. A different approach to depleting SRP from cells is to create a dominant-negative effect through the expression of dominant-negative 7SL RNA mutants. Mutations were introduced into L. collosoma 7SL RNA by inserting a 9-nucleotide linker using the PCR-mega primer method. The mutations were cloned into the pX expression vector together with the upstream tRNAArg that forms the extragenic 7SL RNA promoter (2). We previously demonstrated that L. collosoma 7SL RNA is found in the cell in two conformations, 7SL I and 7SL II (2, 3). The 7SL II form is generated during transcription. Upon binding to the ribosome, 7SL RNA undergoes a conformational change to form 7SL I, which is induced by C-to-U editing (3). The presence of both conformations of 7SL RNA indicates that the SRP is active and can bind ribosomes. Indeed, the ratio of 7SL I to 7SL II changes during growth. In a young culture, which is actively growing and translocating proteins, the level of 7SL I is higher than that of 7SL II, whereas in stationary phase, when growth ceases, the ratio of 7SL I to 7SL II approaches 1:1 (2, 3). These two conformations are very stable and cannot be interconverted in vitro (2). However, separation of the RNA under extremely denaturing conditions in gels containing 75% formamide and 7 M urea results in a single band, suggesting that these molecules can be completely denatured only under severe denaturation conditions (2). We previously showed that nonfunctional 7SL RNA does not undergo the conformational change, and we therefore expect our mutants to be found in a single conformation (3).

To identify mutations that inactivate the function of 7SL RNA, mutations were introduced into each domain of 7SL RNA (Fig. 2A). The expression of 7SL RNA was examined in young cultures of the transgenic parasites (Fig. 2B). RNA was extracted from the transgenic parasites, and the levels of wild-type and tagged 7SL RNAs were examined. The blot was hybridized with an oligonucleotide complementary to the 3' end of 7SL RNA, which recognizes both the mutated and the wild-type 7SL RNA forms (Fig. 2B). The expression of each tagged molecule was also examined by hybridization with a mutation-specific probe (data not shown). The results indicate that all mutants were efficiently expressed in the transgenic parasites. The size of the tagged transcript was proportional to the size of the linker inserted, except for the A3 mutant. The tagged A1, A4, and A7 mutants, each of which carried a 9-nucleotide linker, migrated to the same position on the denaturing gel; whereas the A2 mutant, which has a longer linker, migrated more slowly. Interestingly, the A3 mutant, carrying a linker situated at domain III in the stem opposite the editing site, migrated faster on the same gel, suggesting that this domain may have a special effect on the conformational changes that 7SL RNA undergoes. The presence of each of the tagged molecules in a single conformation suggests that interfering with the proper folding of the molecule by inserting linkers at essential domains compromised the ability of 7SL RNA to undergo its conformational change and that these mutated 7SL RNAs are not functional.


Figure 2
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  		FIG. 2. (A) Schematic representation of the mutations introduced into 7SL RNA. The sequence and secondary structure are as described by Ben-Shlomo et al. (2). The positions at which the mutations were inserted are indicated, and the sequences of the inserts are given. (B) Northern blot analysis showing the levels of 7SL RNAs in cell lines carrying tagged 7SL RNA. RNA (10 µg) was separated on a 10% denaturing gel, electroblotted, and hybridized with an antisense oligonucleotide to 7SL RNA. The positions of wild-type (WT) 7SL I and 7SL II are indicated. The blot was hybridized with 5.8S rRNA to control for the amount of RNA in each sample. The RNA samples are from WT cells, WT-pX cells (carrying WT 7SL RNA cloned into the pX plasmid), and the different 7SL RNA mutant cell lines (indicated by the name of the mutation). (C) Growth rates of WT cells and cells carrying 7SL RNA mutations. The growth of WT cells (solid diamonds) was compared to that of cells carrying the A2 (shaded triangles) or A4 (shaded squares) 7SL RNA mutation. Differential interference contrast images of the cell lines are shown. Bars, 20 µm.

 
The tagged molecules were highly expressed on the multicopy pX plasmid. As was observed previously, the level of 7SL RNA cannot be increased in the cell, most probably because of the limited availability of its binding proteins (2). The highly expressed tagged molecules, therefore, were dominant over the wild-type 7SL RNA expressed from a single-copy gene. For reasons that are not fully understood, not all mutated 7SL RNAs were able to compete with the wild-type transcript with the same efficiency, despite the fact that all were efficiently expressed. This is especially apparent for the A3 and A7 mutants.

To correlate the defects in the level of 7SL RNA with growth, the growth rate of wild-type cells was compared with that of transgenic parasites expressing either the A2 or the A4 mutation. These mutations were chosen for this analysis because only trace amounts of wild-type 7SL RNA (5% of normal levels) were detected in these cells. The results (Fig. 2C) show that cells expressing mutated 7SL RNA grow more slowly. We also noticed that cells expressing mutated 7SL RNA cannot form "rosettes." In the transgenic parasites, the regular structure of "rosettes" is never formed, although we do see interacting cells.

Expression of SP-containing proteins in cell lines expressing the dominant-negative 7SL RNA mutants. To investigate whether SP-containing proteins are affected in cells expressing the 7SL RNA mutants, the biogenesis of a GFP fusion protein containing an SP and a single TM domain (SP-GFP-TM) was examined. The SP and the TM domain were derived from Leishmania donovani 3'/3' nucleotidase/nuclease (7). It was reported previously that overproduction of this protein in Leishmania results in its accumulation not on the surface of the parasite, as expected, but rather in the multivesicular tubule (MVT)-lysosome (10). Overproduction of this protein saturates the trafficking machinery, leading to its degradation within the endosome-lysosome compartment. Although this reporter protein is mislocalized, it is normally translocated across the ER. This protein, therefore, can be used to monitor changes in transport across the ER under conditions of SRP depletion. Cells expressing the A2 or A4 7SL RNA mutation were generated using the pX-hygro plasmid (16). These cells were further used to obtain transgenic parasites also expressing SP-GFP-TM in pX-neo. Cells expressing both plasmids were selected at an elevated G418 concentration. RNA was prepared from these parasites and subjected to Northern blot analysis with a 7SL RNA-specific probe. The results suggest that the level of tagged 7SL RNA remained constant despite expression of another pX plasmid, and clear repression of the wild-type 7SL RNA was observed (Fig. 3A1), suggesting that the dominant-negative phenotype is maintained in these organisms.


Figure 3
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  		FIG. 3. (A1) Level of 7SL RNA in cells carrying the SP-GFP-TM reporter. RNA was prepared from wild-type (WT) cells and from cells carrying the 7SL RNA A2 or A4 mutant on pX-hygro; all cells also carried SP-GFP-TM on the pX-neo plasmid. RNA was separated on a denaturing gel and subjected to Northern blot analysis with an antisense oligonucleotide to 7SL RNA. The positions of 7SL I and 7SL II are indicated. (A2) Biochemical fractionation of SP-GFP-TM. Membrane and soluble fractions were prepared from 109 cells, and aliquots were separated on 12% SDS-polyacrylamide gels and subjected to Western blot analysis with anti-GFP antibodies. The identities of the cell lines used for preparing the samples are indicated. (A3) Confocal microscopy of cells expressing SP-GFP-TM. Cells were treated with FM-4-64 for 40 min, immobilized in agarose, and visualized by confocal microscopy. The images presented, from left to right, are as follows: cells treated with FM-4-64 for 40 min, GFP fluorescence, merging of the FM-4-64 and GFP images, and merging of the FM4-64, GFP, and DIC images. The cell types are given on the left. L, lysosome; E, endosome. (B) The level of BiP does not change in cells carrying 7SL RNA mutants. (B1) Proteins (20 µg) from WT cells and cells carrying the A4 or A2 mutation were subjected to Western blot analysis with anti-BiP antibodies. Reactivity with anti-hnRNPD0 antibodies was used to control for equal loading. (B2) Cells were fixed with 4% (vol/vol) formaldehyde for 25 min and incubated with anti-BiP antibodies, which were detected by an FITC-conjugated secondary antibody. Cells were visualized by confocal microscopy. Nuclei were stained with DAPI. DIC, differential interference contrast; IFA, immunofluorescence assay. Scale bars, 5 µm.

 
After it was verified that the cells carrying the 7SL RNA mutations properly expressed the mRNA for SP-GFP-TM (data not shown), the localization and amount of the protein were examined by biochemical fractionation and confocal microscopy. Cells were lysed by a buffer containing Triton X-100, and membrane-bound and soluble proteins were separated and subjected to Western blot analysis with anti-GFP antibodies. The full-length GFP fusion protein could not be detected in the membrane fraction, and a proteolytically cleaved prGFP was found only in the soluble fraction (Fig. 3A2). Such cleaved GFP (prGFP) was first discovered in the Leishmania MVT-lysosome (8, 29). These data suggest that in wild type cells, the majority of the protein traverses the ER but is mislocalized and is not membrane associated. Note that the fractionation method used separates plasma membrane proteins from soluble proteins, which include proteins extracted from other membrane compartments. The biochemical data are supported by the fluorescence imaging of the cells expressing SP-GFP-TM. Cells were treated with the dye FM-4-64, which stains the acidic endosome and lysosome red. Yellow staining, resulting from merging of the green GFP and the red FM-4-64, was observed, demonstrating that the majority of the protein was localized to the lysosome (Fig. 3A3). These data suggest that despite defects in the SRP pathway, SP-GFP-TM was efficiently translocated to the ER, as in wild-type cells, saturating the trafficking machinery and directing the protein for degradation in the lysosome. This suggests that the SP-GFP-TM protein most probably does not utilize the SRP pathway for translocation but is translocated by the alternative posttranslational pathway.

We next examined the level of the ER chaperone BiP, an SP containing protein, (Fig. 3B1 and 2), in these SRP mutants. The results demonstrate no disruption in the level or localization of this protein, thus supporting the notion that the translocation of SP-containing proteins is unaffected in cells expressing the dominant-negative 7SL RNA mutants.

Expression and trafficking of polytopic membrane proteins in cells expressing the dominant-negative 7SL RNA mutants. As a model for a classical polytopic membrane protein, we chose GFP fused to the glucose transporter (GT3), which is composed of 12 TM domains (6). The same cells carrying the 7SL RNA mutations used for Fig. 3 were used to obtain transgenic parasites expressing the GT3-GFP fusion protein. The localization and amount of the protein were examined by biochemical fractionation and confocal microscopy. As discussed above, overproduction of the GT3-GFP protein in wild-type cells also saturated the trafficking machinery, directing GT3-GFP to degradation in the lysosome. However, the results in Fig. 4A1 and 2 demonstrate major differences between wild-type cells and cells carrying the A2 or A4 7SL RNA mutation. In wild-type cells, very little of the fused GT3-GFP (~78 kDa) was found in the membrane fraction. The majority of GFP (prGFP) was detected in the soluble fraction. On the other hand, in cells carrying a 7SL RNA mutation, the intact 78-kDa GT3-GFP was detected in the membrane fraction and no prGFP was detected in the soluble fraction.


Figure 4
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  		FIG. 4. Localization of polytopic membrane proteins in wild-type (WT) cells and cells carrying 7SL RNA mutants. (A1) Western blot analysis of soluble and membrane-bound GT3-GFP. Membrane and soluble fractions were prepared from 109 cells, and aliquots were subjected to Western blot analysis with anti-GFP antibodies. Ponceau staining or reactivity against anti-hnRNPD0 antibodies was used to control for equal loading of the membrane or soluble fraction, respectively. The positions of GT3-GFP and prGFP are indicated. (A2) GFP fluorescence in WT cells and 7SL RNA mutants carrying pX-GT3-GFP. Cells were fixed in agarose and treated with FM-4-64 for 40 min. The images are as described in the legend to Fig. 3A3. L, lysosome; E, endosome; FP, flagellar pocket. (B) Level and localization of VH+ppase in WT cells and cells carrying 7SL RNA mutants. (B1) Proteins (20 µg) from WT cells and cells carrying the A4 or A2 mutation were subjected to Western blot analysis with anti-VH+ppase antibodies. Reactivity against anti-hnRNPD0 antibodies was used to control for equal loading. (B2) Cells were fixed with 4% (vol/vol) formaldehyde for 25 min and incubated with anti-VH+ppase antibodies, which were detected by an FITC-conjugated secondary antibody. Cells were visualized by confocal microscopy. Nuclei were stained with DAPI. DIC, differential interference contrast; IFA, immunofluorescence assay. Scale bars, 5 µm.

 
We next examined the localization of the protein by confocal microscopy. The results presented in Fig. 4A2 demonstrate completely different localizations for wild-type and mutant cell lines. Whereas in the wild-type cells, the protein was mostly colocalized with FM-4-64 in the lysosome, in the mutant cells, the protein was localized either in the flagellar pocket or in the plasma membrane. Note that all proteins destined for plasma membrane localization first traverse the flagellar pocket. Importantly, there was no colocalization of the GFP fusion protein with the FM-4-64 dye in the mutant cells, suggesting that in these cells, GT3-GFP is not destined for degradation. Thus, these data demonstrate major differences in the fates of SP-GFP and GT3-GFP in the mutant cells. We next examined the expression of the endogenous membrane protein VH+ppase in the acidocalcisome. The results presented in Fig. 4B1 suggest that the level of acidocalcisome VH+ppase was reduced by 45% in cell lines with mutated 7SL RNA. Immunofluorescence of this protein inside the acidocalcisomes shows a reduction in the number of these organelles containing the protein (Fig. 4B2).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, the biogenesis of membrane and SP-containing proteins was examined in two trypanosomatid species under conditions of SRP limitation, achieved either by RNAi silencing of SRP proteins in T. brucei or by overexpression of dominant-negative 7SL RNA mutants in L. collosoma. The translocation of polytopic membrane proteins, but not of SP-containing proteins, across the ER membrane was perturbed in these SRP-deficient cells, suggesting that polytopic membrane proteins are preferential substrates of the SRP pathway.

We previously examined the effect of SRP limitation on the biogenesis of SP-containing proteins by depleting the T. brucei SRP pathway through silencing of the SRP54 protein. This depletion has no effect on the levels of these proteins or their entry to the ER, but the proteins are mislocalized (24). We proposed that the mislocalization might originate from defects in polytopic membrane protein biogenesis. In this study, we demonstrated that, indeed, the primary defect due to SRP depletion is in the biogenesis of membrane proteins. This was demonstrated both for GFP-fused transporters and for endogenous proteins carrying TM domains. Similar results were observed for two trypanosome species by using two different approaches to deplete the SRP. Based on these data, we propose that in trypanosomes, as in E. coli, SRP may exclusively translocate polytopic membrane proteins (35, 37).

Most recent studies examined the translocation of both polytopic membrane proteins and SP-containing proteins upon RNAi silencing of SRP proteins in cultured mammalian cells. Interestingly, and in contrast to the results reported here, the levels of both SP-containing proteins and TM proteins were reduced in the silenced cells, suggesting that in mammals, SRP translocates both types of proteins. Since SRP depletion was not complete in the silenced mammalian cells, the study examined the effect of SRP limitation on protein translocation. In these mammalian cells, polytopic membrane proteins were less perturbed than SP-containing proteins, most probably because these proteins have higher affinity for the SRP and can compete with SP-containing proteins, which have lower affinity for the SRP (19). Collectively, these results may suggest that the primordial function of the SRP was to translocate polytopic membrane proteins and that only later in evolution was the SRP adjusted to translocate SP-containing proteins as well. Trypanosomes, however, seem to differ from E. coli and even from other eukaryotes in being promiscuous in their choice of the pathway for translocation of SP-containing proteins. In a recent study, we demonstrated that during RNAi silencing of SEC71, a factor that belongs exclusively to the posttranslational translocation pathway enabled the translocation of SP-containing proteins to the ER, suggesting that trypanosomes can interchangeably utilize either route for the translocation of SP-containing proteins across the ER (H. Goldshmidt et al., submitted for publication). In contrast, in the yeast S. cerevisiae, there is a clear commitment of a protein to its translocation route, and if this route is defective, the protein will not be translocated by the alternative pathway (5, 13, 36).

The reductions in the levels of proteins carrying multiple TM domains (nucleobase transporter, TB29-2, and VH+ppase) upon SRP depletion seem to be direct effects of this perturbation. It is easy to comprehend that in the absence of SRP, these proteins could not be properly translocated and hence might be exposed to proteolysis. In mammals, treatment of SRP-silenced cells with the proteasome inhibitor MG132 increases the level of intracellular protein, which is normally secreted from the cell. However, no effect of this inhibitor on the level of preprotein or on membrane proteins was reported (19). Treatment of T. brucei SRP68-silenced cells with lactacystin did not affect the level of VH+ppase, suggesting that the proteasome is not involved in this degradation (Fig. 1Da). Proteolysis also was not inhibited by the lysosome inhibitor (Fig. 1Db) bafilomycin, suggesting that this proteolysis does not take place in the lysosome but is most probably mediated by an as yet unidentified protease, which might be localized on the face of the ER membrane. In recent years, different types of intramembrane proteases have been described (41), and such proteases may also function to degrade misfolded proteins that accumulate on the ER membrane as a result of mistargeting by the SRP. However, we cannot exclude the possibility that the failure to detect the misfolded proteins is due to the fact these form aggregates that are not solubilized by the detergents used.

The finding that nascent synthesis of VH+ppase can be detected by in vivo labeling experiments (Fig. 1E) suggests that polytopic membrane proteins are synthesized during SRP depletion. Indeed, we recently demonstrated that the levels of mRNAs, including VH+ppase mRNA, are not changed when SRP proteins are depleted (26). The steady-state reductions in the levels of the polytopic membrane proteins observed in this study do not result from destabilization of these mRNAs or from specific translation inhibition of polytopic membrane proteins but rather reflect either the proteolysis or the aggregation of these misfolded proteins.

The effect of SRP depletion on GT3-GFP in L. collosoma is complex compared to the effect observed for the TbNT8.1 transporter in T. brucei. Whereas in T. brucei, the transporter was not detected under conditions of SRP depletion, in L. collosoma the protein was able to traverse the ER membrane. These differences reflect the level of depletion, which is much more complete by RNAi. The SRP deficiency in L. collosoma "helped" the cells to properly localize the GT3-GFP. Under normal conditions, the secretory pathway is overloaded with this protein, and the protein is therefore destined for degradation. However, under SRP depletion, attenuation of the translocation to the ER reduces the overload, thus enabling the protein to reach its correct destination, the plasma membrane. The localization of SP-GFP-TM and GT3-GFP observed in this study resembles the mislocalization observed in Leishmania (10). Overexpression of the SP-GFP-TM fusion protein in Leishmania led to its accumulation in the MVT-lysosome (10). It was proposed that the anterograde trafficking of this chimeric protein to the cell surface occurs by vesicular traffic from the Golgi apparatus to the flagellar reservoir. However, when the protein is overexpressed, the excess GFP fusion proteins are shunted from the Golgi apparatus into vesicles fated for fusion directly to the MVT-lysosome, without reaching the flagellar pocket. This explains why we could never detect either GFP-GT3 or SP-GFP-TM in the flagellar pocket or on the cell surface in wild-type cells. However, when SRP was limited, enabling only a limited amount of GT3-GFP to traverse the membrane, the protein was not shunted for degradation but was directed to the flagellar pocket and the plasma membrane. In contrast, SP-GFP-TM was mostly localized to the lysosome even under SRP depletion, suggesting that the majority of this protein traverses the ER by the alternative posttranslational pathway, saturates the intracellular trafficking system, and is therefore destined for degradation.

Nothing is known about how multi-TM proteins, such as the VH+ppase, are targeted to the acidocalcisome. This study provides evidence, for the first time, that translocation of proteins to the acidocalcisomes is mediated by the SRP in both T. brucei and L. collosoma. Decreases in the amount and localization of the protein in acidocalcisomes were observed under conditions of SRP depletion, suggesting that this protein is an SRP substrate. It is not known, however, whether the protein reaches the acidocalcisome from the Golgi apparatus or whether the protein is delivered to this organelle from the plasma membrane.

In sum, this study demonstrates that polytopic membrane proteins are translocated to the ER via the SRP pathway, whereas SP-containing proteins can utilize an alternative translocation pathway. This differential choice in trypanosomes resembles the translocation of proteins in bacteria more closely than that in mammalian cells.


   ACKNOWLEDGMENTS
 
This research was supported in part by grant 611/00-1 from the Israel Science Foundation. S.M. is an international scholar of the Howard Hughes Medical Institute. S.M. holds the David and Inez Myers Chair in RNA silencing of diseases.


   FOOTNOTES
 
* Corresponding author. Mailing address: The Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat Gan 52900, Israel. Phone: 972-3-5318068. Fax: 972-37384058. E-mail: michaes{at}mail.biu.ac.il Back

{triangledown} Published ahead of print on 22 August 2007. Back

{dagger} Y.L., Y.V., and H.G. contributed equally to this study. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Béjà, O., E. Ullu, and S. Michaeli. 1993. Identification of a tRNA-like molecule that copurifies with the 7SL RNA of Trypanosoma brucei. Mol. Biochem. Parasitol. 57:223-229.[CrossRef][Medline]
  2. Ben-Shlomo, H., A. Levitan, O. Béjà, and S. Michaeli. 1997. The trypanosomatid Leptomonas collosoma 7SL RNA gene. Analysis of elements controlling its expression. Nucleic Acids Res. 25:4977-4984.[Abstract/Free Full Text]
  3. Ben-Shlomo, H., A. Levitan, N. E. Shay, I. Goncharov, and S. Michaeli. 1999. RNA editing associated with the generation of two distinct conformations of the trypanosomatid Leptomonas collosoma 7SL RNA. J. Biol. Chem. 274:25642-25650.[Abstract/Free Full Text]
  4. Besteiro, S., R. A. Williams, L. S. Morrison, G. H. Coombs, and J. C. Mottram. 2006. Endosome sorting and autophagy are essential for differentiation and virulence of Leishmania major. J. Biol. Chem. 281:11384-11396.[Abstract/Free Full Text]
  5. Brown, J. D., B. C. Hann, K. F. Medzihradszky, M. Niwa, A. L. Burlingame, and P. Walter. 1994. Subunits of the Saccharomyces cerevisiae signal recognition particle required for its functional expression. EMBO J. 13:4390-4400.[Medline]
  6. Burchmore, R. J., and S. M. Landfear. 1998. Differential regulation of multiple glucose transporter genes in Leishmania mexicana. J. Biol. Chem. 273:29118-29126.[Abstract/Free Full Text]
  7. Debrabant, A., E. Ghedin, and D. M. Dwyer. 2000. Dissection of the functional domains of the Leishmania surface membrane 3'-nucleotidase/nuclease, a unique member of the class I nuclease family. J. Biol. Chem. 275:16366-16372.[Abstract/Free Full Text]
  8. Dodge, M. A., R. F. Waller, L. M. Chow, M. M. Zaman, L. M. Cotton, M. J. McConville, and D. F. Wirth. 2004. Localization and activity of multidrug resistance protein 1 in the secretory pathway of Leishmania parasites. Mol. Microbiol. 51:1563-1575.[CrossRef][Medline]
  9. Egea, P. F., R. M. Stroud, and P. Walter. 2005. Targeting proteins to membranes: structure of the signal recognition particle. Curr. Opin. Struct. Biol. 15:213-220.[CrossRef][Medline]
  10. Ghedin, E., A. Debrabant, J. C. Engel, and D. M. Dwyer. 2001. Secretory and endocytic pathways converge in a dynamic endosomal system in a primitive protozoan. Traffic 2:175-188.[CrossRef][Medline]
  11. Gilmore, R., P. Walter, and G. Blobel. 1982. Protein translocation across the endoplasmic reticulum. II. Isolation and characterization of the signal recognition particle receptor. J. Cell Biol. 95:470-477.[Abstract/Free Full Text]
  12. Goldring, A., Y. Zimmer, E. Ben Yehuda, I. Goncharov, and S. Michaeli. 1996. Stable transfection in the monogenetic trypanosomatid Leptomonas collosoma—transcription barrier of heterologous trypanosomatid SL RNA genes and expression of a chimeric SL RNA molecule. Exp. Parasitol. 84:28-41.[CrossRef][Medline]
  13. Hann, B. C., and P. Walter. 1991. The signal recognition particle in S. cerevisiae. Cell 67:131-144.[CrossRef][Medline]
  14. Henriques, C., M. A. Sanchez, R. Tryon, and S. M. Landfear. 2003. Molecular and functional characterization of the first nucleobase transporter gene from African trypanosomes. Mol. Biochem. Parasitol. 130:101-110.[CrossRef][Medline]
  15. Hill, J. E., D. A. Scott, S. Luo, and R. Docampo. 2000. Cloning and functional expression of a gene encoding a vacuolar-type proton-translocating pyrophosphatase from Trypanosoma cruzi. Biochem. J. 351:281-288.[CrossRef][Medline]
  16. Kapler, G. M., C. M. Coburn, and S. M. Beverley. 1990. Stable transfection of the human parasite Leishmania major delineates a 30-kilobase region sufficient for extrachromosomal replication and expression. Mol. Cell. Biol. 10:1084-1094.[Abstract/Free Full Text]
  17. Keenan, R. J., D. M. Freymann, R. M. Stroud, and P. Walter. 2001. The signal recognition particle. Annu. Rev. Biochem. 70:755-775.[CrossRef][Medline]
  18. Koch, H. G., M. Moser, and M. Muller. 2003. Signal recognition particle-dependent protein targeting, universal to all kingdoms of life. Rev. Physiol. Biochem. Pharmacol. 146:55-94.[Medline]
  19. Lakkaraju, A. K., P. P. Luyet, P. Parone, T. Falguieres, and K. Strub. 2007. Inefficient targeting to the endoplasmic reticulum by the signal recognition particle elicits selective defects in post-ER membrane trafficking. Exp. Cell Res. 313:834-847.[CrossRef][Medline]
  20. Lee, H. C., and H. D. Bernstein. 2001. The targeting pathway of Escherichia coli presecretory and integral membrane proteins is specified by the hydrophobicity of the targeting signal. Proc. Natl. Acad. Sci. USA 98:3471-3476.[Abstract/Free Full Text]
  21. Lee, M. G., B. E. Bihain, D. G. Russell, R. J. Deckelbaum, and L. H. Van der Ploeg. 1990. Characterization of a cDNA encoding a cysteine-rich cell surface protein located in the flagellar pocket of the protozoan Trypanosoma brucei. Mol. Cell. Biol. 10:4506-4517.[Abstract/Free Full Text]
  22. Lee, M. G., D. G. Russell, P. A. D'Alesandro, and L. H. Van der Ploeg. 1994. Identification of membrane-associated proteins in Trypanosoma brucei encoding an internal, EARLRAEE amino acid repeat. J. Biol. Chem. 269:8408-8415.[Abstract/Free Full Text]
  23. Liu, L., H. Ben-Shlomo, Y. X. Xu, M. Z. Stern, I. Goncharov, Y. Zhang, and S. Michaeli. 2003. The trypanosomatid signal recognition particle consists of two RNA molecules, a 7SL RNA homologue and a novel tRNA-like molecule. J. Biol. Chem. 278:18271-18280.[Abstract/Free Full Text]
  24. Liu, L., X. H. Liang, S. Uliel, R. Unger, E. Ullu, and S. Michaeli. 2002. RNA interference of signal peptide-binding protein SRP54 elicits deleterious effects and protein sorting defects in trypanosomes. J. Biol. Chem. 277:47348-47357.[Abstract/Free Full Text]
  25. Lustig, Y., H. Goldshmidt, S. Uliel, and S. Michaeli. 2005. The Trypanosoma brucei signal recognition particle lacks the Alu-domain-binding proteins: purification and functional analysis of its binding proteins by RNAi. J. Cell Sci. 118:4551-4562.[Abstract/Free Full Text]
  26. Lustig, Y., L. Sheiner, Y. Vagima, H. Goldshmidt, A. Das, V. Bellofatto, and S. Michaeli. 2007. Spliced-leader RNA silencing: a novel stress-induced mechanism in Trypanosoma brucei. EMBO Rep. 8:408-413.[CrossRef][Medline]
  27. Meacock, S. L., J. J. Greenfield, and S. High. 2000. Protein targeting and translocation at the endoplasmic reticulum membrane—through the eye of a needle? Essays Biochem. 36:1-13.[Medline]
  28. Müller, M., H. G. Koch, K. Beck, and U. Schafer. 2001. Protein traffic in bacteria: multiple routes from the ribosome to and across the membrane. Prog. Nucleic Acid Res. Mol. Biol. 66:107-157.[Medline]
  29. Mullin, K. A., B. J. Foth, S. C. Ilgoutz, J. M. Callaghan, J. L. Zawadzki, G. I. McFadden, and M. J. McConville. 2001. Regulated degradation of an endoplasmic reticulum membrane protein in a tubular lysosome in Leishmania mexicana. Mol. Biol. Cell 12:2364-2377.[Abstract/Free Full Text]
  30. Mutka, S. C., and P. Walter. 2001. Multifaceted physiological response allows yeast to adapt to the loss of the signal recognition particle-dependent protein-targeting pathway. Mol. Biol. Cell 12:577-588.[Abstract/Free Full Text]
  31. Ng, D. T., J. D. Brown, and P. Walter. 1996. Signal sequences specify the targeting route to the endoplasmic reticulum membrane. J. Cell Biol. 134:269-278.[Abstract/Free Full Text]
  32. Pool, M. R., J. Stumm, T. A. Fulga, I. Sinning, and B. Dobberstein. 2002. Distinct modes of signal recognition particle interaction with the ribosome. Science 297:1345-1348.[Abstract/Free Full Text]
  33. Poritz, M. A., H. D. Bernstein, K. Strub, D. Zopf, H. Wilhelm, and P. Walter. 1990. An E. coli ribonucleoprotein containing 4.5S RNA resembles mammalian signal recognition particle. Science 250:1111-1117.[Abstract/Free Full Text]
  34. Ren, Y. G., K. W. Wagner, D. A. Knee, P. Aza-Blanc, M. Nasoff, and Q. L. Deveraux. 2004. Differential regulation of the TRAIL death receptors DR4 and DR5 by the signal recognition particle. Mol. Biol. Cell 15:5064-5074.[Abstract/Free Full Text]
  35. Seluanov, A., and E. Bibi. 1997. FtsY, the prokaryotic signal recognition particle receptor homologue, is essential for biogenesis of membrane proteins. J. Biol. Chem. 272:2053-2055.[Abstract/Free Full Text]
  36. Stirling, C. J., and E. W. Hewitt. 1992. The S. cerevisiae SEC65 gene encodes a component of yeast signal recognition particle with homology to human SRP19. Nature 356:534-537.[CrossRef][Medline]
  37. Ulbrandt, N. D., J. A. Newitt, and H. D. Bernstein. 1997. The E. coli signal recognition particle is required for the insertion of a subset of inner membrane proteins. Cell 88:187-196.[CrossRef][Medline]
  38. Walter, P., and G. Blobel. 1981. Translocation of proteins across the endoplasmic reticulum. III. Signal recognition protein (SRP) causes signal sequence-dependent and site-specific arrest of chain elongation that is released by microsomal membranes. J. Cell Biol. 91:557-561.[Abstract/Free Full Text]
  39. Walter, P., I. Ibrahimi, and G. Blobel. 1981. Translocation of proteins across the endoplasmic reticulum. I. Signal recognition protein (SRP) binds to in-vitro-assembled polysomes synthesizing secretory protein. J. Cell Biol. 91:545-550.[Abstract/Free Full Text]
  40. Walter, P., and A. E. Johnson. 1994. Signal sequence recognition and protein targeting to the endoplasmic reticulum membrane. Annu. Rev. Cell Biol. 10:87-119.[CrossRef][Medline]
  41. Wolfe, M. S., and R. Kopan. 2004. Intramembrane proteolysis: theme and variations. Science 305:1119-1123.[Abstract/Free Full Text]
  42. Wolin, S. L., and P. Walter. 1989. Signal recognition particle mediates a transient elongation arrest of preprolactin in reticulocyte lysate. J. Cell Biol. 109:2617-2622.[Abstract/Free Full Text]
  43. Yoshimori, T., A. Yamamoto, Y. Moriyama, M. Futai, and Y. Tashiro. 1991. Bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells. J. Biol. Chem. 266:17707-17712.[Abstract/Free Full Text]

Eukaryotic Cell, October 2007, p. 1865-1875, Vol. 6, No. 10
1535-9778/07/$08.00+0     doi:10.1128/EC.00134-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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