Human-Like Eukaryotic Translation Initiation Factor 3 from Neurospora crassa

Eukaryotic translation initiation factor 3 (eIF3) is a key regulator of translation initiation, but its in vivo assembly and molecular functions remain unclear. Here we show that eIF3 from Neurospora crassa is structurally and compositionally similar to human eIF3. N. crassa eIF3 forms a stable 12-subunit complex linked genetically and biochemically to the 13^th subunit, eIF3j, which in humans modulates mRNA start codon selection. Based on N. crassa genetic analysis, most subunits in eIF3 are essential. Subunits that can be deleted (e, h, k and l) map to the right side of the eIF3 complex, suggesting that they may coordinately regulate eIF3 function. Consistent with this model, subunits eIF3k and eIF3l are incorporated into the eIF3 complex as a pair, and their insertion depends on the presence of subunit eIF3h, a key regulator of vertebrate development. Comparisons to other eIF3 complexes suggest that eIF3 assembles around an eIF3a and eIF3c dimer, which may explain the coordinated regulation of human eIF3 levels. Taken together, these results show that Neurospora crassa eIF3 provides a tractable system for probing the structure and function of human-like eIF3 in the context of living cells.     

Trabecular architecture and joint loading of the proximal humerus in extant hominoids,Ateles, and Australopithecus africanus

Objectives

Several studies have investigated potential functional signals in the trabecular structure of the primate proximal humerus but with varied success. Here, we apply for the first time a “whole‐epiphyses” approach to analysing trabecular bone in the humeral head with the aim of providing a more holistic interpretation of trabecular variation in relation to habitual locomotor or manipulative behaviors in several extant primates and Australopithecus africanus.

Materials and methods

We use a “whole‐epiphysis” methodology in comparison to the traditional volume of interest (VOI) approach to investigate variation in trabecular structure and joint loading in the proximal humerus of extant hominoids, Ateles and A. africanus (StW 328).

Results

There are important differences in the quantification of trabecular parameters using a “whole‐epiphysis” versus a VOI‐based approach. Variation in trabecular structure across knuckle‐walking African apes, suspensory taxa, and modern humans was generally consistent with predictions of load magnitude and inferred joint posture during habitual behaviors. Higher relative trabecular bone volume and more isotropic trabeculae in StW 328 suggest A. africanus may have still used its forelimbs for arboreal locomotion.

Discussion

A whole‐epiphysis approach to analysing trabecular structure of the proximal humerus can help distinguish functional signals of joint loading across extant primates and can provide novel insight into habitual behaviors of fossil hominins.

     

Evidence of Combat in Triceratops

Background

The horns and frill of Triceratops and other ceratopsids (horned dinosaurs) are interpreted variously as display structures or as weapons against conspecifics and predators. Lesions (in the form of periosteal reactive bone, healing fractures, and alleged punctures) on Triceratops skulls have been used as anecdotal support of intraspecific combat similar to that in modern horned and antlered animals. If ceratopsids with different cranial morphologies used their horns in such combat, this should be reflected in the rates of lesion occurrence across the skull.

Methodology/Principal Findings

We used a G-test of independence to compare incidence rates of lesions in Triceratops (which possesses two large brow horns and a smaller nasal horn) and the related ceratopsid Centrosaurus (with a large nasal horn and small brow horns), for the nasal, jugal, squamosal, and parietal bones of the skull. The two taxa differ significantly in the occurrence of lesions on the squamosal bone of the frill (P = 0.002), but not in other cranial bones (P>0.20).

Conclusions/Significance

This pattern is consistent with Triceratops using its horns in combat and the frill being adapted as a protective structure for this taxon. Lower pathology rates in Centrosaurus may indicate visual rather than physical use of cranial ornamentation in this genus, or a form of combat focused on the body rather than the head.     

Evidence for a dual pattern of cranial venous sinuses on the endocranial cast of Taung (Australopithecus africanus)

According to published accounts, an enlarged occipital-marginal sinus system is absent in Australopithecus africanus, although it occurs in high frequencies in A. robustus, A. Boisei, and Hadar hominids commonly designated A. afarensis. In this report, we describe, for the first time, an enlarged occipital-marginal sinus system on the endocranial cast of the Taung specimen, which is part of the holotype of A. africanus. In addition, well-developed right transverse and sigmoid sinuses are represented on the Taung endocast. The various components of the dual venous sinus system on the Taung endocast are measured, and the system is compared to those of other fossil hominids. The compresence of a lateral sinus system and enlarged occipital and marginal sinuses occurs in two Hadar specimens, 2 specimens of A. robustus crassidens, 1 A. boisei specimen, and several early H. sapiens crania. Hence, the presence of strong transverse sinus impressions in a fragmentary specimen may not be interpreted as an indication that an enlarged occipital-marginal sinus system was not present in the original specimen. Conversely, lack of transverse sinus grooves in a fragmentary specimen does provide indirect evidence than an enlarged occipital-marginal system would probably have been present in the whole specimen, as in 2 specimens of A. boisei. Including Taung, enlarged occipital and marginal sinuses occur in 1 out of 5, or 20%, of A. africanus specimens. This figure compares well with the range of mean frequencies in modern human cranial series (1.5 to 28%), but is much lower than are the frequencies for A. boisei, A. robustus, and the Hadar hominids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for a Slow-Turnover Form of the Ca2+-Independent Phosphoenolpyruvate Carboxylase Kinase in the Aleurone-Endosperm Tissue of Germinating Barley Seeds

Phosphoenolpyruvate carboxylase (PEPC) activity was detected in aleurone-endosperm extracts of barley (Hordeum vulgare) seeds during germination, and specific anti-sorghum (Sorghum bicolor) C₄ PEPC polyclonal antibodies immunodecorated constitutive 103-kD and inducible 108-kD PEPC polypeptides in western analysis. The 103- and 108-kD polypeptides were radiolabeled in situ after imbibition for up to 1.5 d in ³²P-labeled inorganic phosphate. In vitro phosphorylation by a Ca²⁺-independent PEPC protein kinase (PK) in crude extracts enhanced the enzyme''s velocity and decreased its sensitivity to l-malate at suboptimal pH and [PEP]. Isolated aleurone cell protoplasts contained both phosphorylated PEPC and a Ca²⁺-independent PEPC-PK that was partially purified by affinity chromatography on blue dextran-agarose. This PK activity was present in dry seeds, and PEPC phosphorylation in situ during imbibition was not affected by the cytosolic protein-synthesis inhibitor cycloheximide, by weak acids, or by various pharmacological reagents that had proven to be effective blockers of the light signal transduction chain and PEPC phosphorylation in C₄ mesophyll protoplasts. These collective data support the hypothesis that this Ca²⁺-independent PEPC-PK was formed during maturation of barley seeds and that its presumed underlying signaling elements were no longer operative during germination.Higher-plant PEPC (EC 4.1.1.31) is subject to in vivo phosphorylation of a regulatory Ser located in the N-terminal domain of the protein. In vitro phosphorylation by a Ca²⁺-independent, low-molecular-mass (30–39 kD) PEPC-PK modulates PEPC regulation interactively by opposing metabolite effectors (e.g. allosteric activation by Glc-6-P and feedback inhibition by l-malate; Andreo et al., 1987), decreasing significantly the extent of malate inhibition of the leaf enzyme (Carter et al., 1991; Chollet et al., 1996; Vidal et al., 1996; Vidal and Chollet, 1997). These metabolites control the rate of phosphorylation of PEPC via an indirect target-protein effect (Wang and Chollet, 1993; Echevarría et al., 1994; Vidal and Chollet, 1997).Several lines of evidence support the view that this protein-Ser/Thr kinase is the physiologically relevant PEPC-PK (Li and Chollet, 1993; Chollet et al., 1996; Vidal et al., 1996; Vidal and Chollet, 1997). The presence and inducible nature of leaf PEPC-PK have been established further in various C₃, C₄, and CAM plant species (Chollet et al., 1996). In all cases, CHX proved to be a potent inhibitor of this up-regulation process so that apparent changes in the turnover rate of PEPC-PK itself or another, as yet unknown, protein factor were invoked to account for this observation (Carter et al., 1991; Jiao et al., 1991; Chollet et al., 1996). Consistent with this proposal are recent findings about PEPC-PK from leaves of C₃, C₄, and CAM plants that determined activity levels of the enzyme to depend on changes in the level of the corresponding translatable mRNA (Hartwell et al., 1996).Using a cellular approach we previously showed in sorghum (Sorghum bicolor) and hairy crabgrass (Digitaria sanguinalis) that PEPC-PK is up-regulated in C₄ mesophyll cell protoplasts following illumination in the presence of a weak base (NH₄Cl or methylamine; Pierre et al., 1992; Giglioli-Guivarc''h et al., 1996), with a time course (1–2 h) similar to that of the intact, illuminated sorghum (Bakrim et al., 1992) or maize leaf (Echevarría et al., 1990). This light- and weak-base-dependent process via a complex transduction chain is likely to involve sequentially an increase in pHc, inositol trisphosphate-gated Ca²⁺ channels of the tonoplast, an increase in cytosolic Ca²⁺, a Ca²⁺-dependent PK, and PEPC-PK.Considerably less is known about the up-regulation of PEPC-PK and PEPC phosphorylation in nongreen tissues. A sorghum root PEPC-PK purified on BDA was shown to phosphorylate in vitro both recombinant C₄ PEPC and the root C₃-like isoform, thereby decreasing the enzyme''s malate sensitivity (Pacquit et al., 1993). PEPC from soybean root nodules was phosphorylated in vitro and in vivo by an endogenous PK (Schuller and Werner, 1993; Zhang et al., 1995; Zhang and Chollet, 1997). A Ca²⁺-independent nodule PEPC-PK containing two active polypeptides (32–37 kD) catalyzed the incorporation of phosphate on a Ser residue of the target enzyme and was modulated by photosynthate transported from the shoots (Zhang and Chollet, 1997). Regulatory seryl phosphorylation of a heterotetrameric (α₂β₂) banana fruit PEPC by a copurifying, Ca²⁺-independent PEPC-PK was shown to occur in vitro (Law and Plaxton, 1997). Although phosphorylation was also detected in vivo and found to concern primarily the α-subunit, PEPC exists mainly in the dephosphorylated form in preclimacteric, climacteric, and postclimacteric fruit.In a previous study we showed that PEPC undergoes regulatory phosphorylation in aleurone-endosperm tissue during germination of wheat seeds (Osuna et al., 1996). Here we report on PEPC and the requisite PEPC-PK in germinating barley (Hordeum vulgare) seeds. PEPC was highly phosphorylated by a Ca²⁺-independent Ser/Thr PEPC-PK similar to that found in other plant systems studied previously (Chollet et al., 1996); however, the PK was already present in the dry seed and its activity did not require protein synthesis during imbibition.     

Evidence for a Cytoskeleton-Associated Binding Site Involved in Prolamine mRNA Localization to the Protein Bodies in Rice Endosperm Tissue

Previous studies have demonstrated that the mRNAs encoding the prolamine and glutelin storage proteins are localized to morphologically distinct membranes of the endoplasmic reticulum (ER) complex in developing rice (Oryza sativa L.) endosperm cells. To gain insight about this mRNA localization process, we investigated the association of prolamine polysomes on the ER that delimit the prolamine protein bodies (PBs). The bulk of the prolamine polysomes were resistant to extraction by 1% Triton X-100 either alone or together with puromycin, which suggests that these translation complexes are anchored to the PB surface through a second binding site in addition to the well-characterized ribosome-binding site of the ER-localized protein translocation complex. Suppression of translation initiation shows that these polysomes are bound through the mRNA, as shown by the simultaneous increase in the amounts of ribosome-free prolamine mRNAs and decrease in prolamine polysome content associated with the membrane-stripped PB fraction. The prolamine polysome-binding activity is likely to be associated with the cytoskeleton, based on the association of actin and tubulin with the prolamine polysomes and PBs after sucrose-density centrifugation.     

Dimethylsulfoniopropionate Biosynthesis in Spartina alterniflora : Evidence That S-Methylmethionine and Dimethylsulfoniopropylamine Are Intermediates

The osmoprotectant 3-dimethylsulfoniopropionate (DMSP) occurs in Gramineae and Compositae, but its synthesis has been studied only in the latter. The DMSP synthesis pathway was therefore investigated in the salt marsh grass Spartina alterniflora Loisel. Leaf tissue metabolized supplied [³⁵S]methionine (Met) to S-methyl-l-Met (SMM), 3-dimethylsulfoniopropylamine (DMSP-amine), and DMSP. The ³⁵S-labeling kinetics of SMM and DMSP-amine indicated that they were intermediates and, consistent with this, the dimethylsulfonium moiety of SMM was shown by stable isotope labeling to be incorporated as a unit into DMSP. The identity of DMSP-amine, a novel natural product, was confirmed by both chemical and mass-spectral methods. S. alterniflora readily converted supplied [³⁵S]SMM to DMSP-amine and DMSP, and also readily converted supplied [³⁵S]DMSP-amine to DMSP; grasses that lack DMSP did neither. A small amount of label was detected in 3-dimethylsulfoniopropionaldehyde (DMSP-ald) when [³⁵S]SMM or [³⁵S]DMSP-amine was given. These results are consistent with the operation of the pathway Met → SMM → DMSP-amine → DMSP-ald → DMSP, which differs from that found in Compositae by the presence of a free DMSP-amine intermediate. This dissimilarity suggests that DMSP synthesis evolved independently in Gramineae and Compositae.     

Plasma Membrane-Associated Actin in Bright Yellow 2 Tobacco Cells : Evidence for Interaction with Microtubules

Plasma membrane ghosts form when plant protoplasts attached to a substrate are lysed to leave a small patch of plasma membrane. We have identified several factors, including the use of a mildly acidic actin stabilization buffer and the inclusion of glutaraldehyde in the fixative, that allow immunofluorescent visualization of extensive cortical actin arrays retained on membrane ghosts made from tobacco (Nicotiana tabacum L.) suspension-cultured cells (line Bright Yellow 2). Normal microtubule arrays were also retained using these conditions. Membrane-associated actin is random; it exhibits only limited coalignment with the microtubules, and microtubule depolymerization in whole cells before wall digestion and ghost formation has little effect on actin retention. Actin and microtubules also exhibit different sensitivities to the pH and K⁺ and Ca²⁺ concentrations of the lysis buffer. There is, however, strong evidence for interactions between actin and the microtubules at or near the plasma membrane, because both ghosts and protoplasts prepared from taxol-pretreated cells have microtubules arranged in parallel arrays and an increased amount of actin coaligned with the microtubules. These experiments suggest that the organization of the cortical actin arrays may be dependent on the localization and organization of the microtubules.     

Kinematic Validation of a Multi-Kinect v2 Instrumented 10-Meter Walkway for Quantitative Gait Assessments

Walking ability is frequently assessed with the 10-meter walking test (10MWT), which may be instrumented with multiple Kinect v2 sensors to complement the typical stopwatch-based time to walk 10 meters with quantitative gait information derived from Kinect’s 3D body point’s time series. The current study aimed to evaluate a multi-Kinect v2 set-up for quantitative gait assessments during the 10MWT against a gold-standard motion-registration system by determining between-systems agreement for body point’s time series, spatiotemporal gait parameters and the time to walk 10 meters. To this end, the 10MWT was conducted at comfortable and maximum walking speed, while 3D full-body kinematics was concurrently recorded with the multi-Kinect v2 set-up and the Optotrak motion-registration system (i.e., the gold standard). Between-systems agreement for body point’s time series was assessed with the intraclass correlation coefficient (ICC). Between-systems agreement was similarly determined for the gait parameters’ walking speed, cadence, step length, stride length, step width, step time, stride time (all obtained for the intermediate 6 meters) and the time to walk 10 meters, complemented by Bland-Altman’s bias and limits of agreement. Body point’s time series agreed well between the motion-registration systems, particularly so for body points in motion. For both comfortable and maximum walking speeds, the between-systems agreement for the time to walk 10 meters and all gait parameters except step width was high (ICC ≥ 0.888), with negligible biases and narrow limits of agreement. Hence, body point’s time series and gait parameters obtained with a multi-Kinect v2 set-up match well with those derived with a gold standard in 3D measurement accuracy. Future studies are recommended to test the clinical utility of the multi-Kinect v2 set-up to automate 10MWT assessments, thereby complementing the time to walk 10 meters with reliable spatiotemporal gait parameters obtained objectively in a quick, unobtrusive and patient-friendly manner.     

In Vivo Evidence for Platelet-Induced Physiological Angiogenesis by a COX Driven Mechanism

We sought to determine a role for platelets in in vivo angiogenesis, quantified by changes in the capillary to fibre ratio (C∶F) of mouse skeletal muscle, utilising two distinct forms of capillary growth to identify differential effects. Capillary sprouting was induced by muscle overload, and longitudinal splitting by chronic hyperaemia. Platelet depletion was achieved by anti-GPIbα antibody treatment. Sprouting induced a significant increase in C∶F (1.42±0.02 vs. contralateral 1.29±0.02, P<0.001) that was abolished by platelet depletion, while the significant C∶F increase caused by splitting (1.40±0.03 vs. control 1.28±0.03, P<0.01) was unaffected. Granulocyte/monocyte depletion showed this response was not immune-regulated. VEGF overexpression failed to rescue angiogenesis following platelet depletion, suggesting the mechanism is not simply reliant on growth factor release. Sprouting occurred normally following antibody-induced GPVI shedding, suggesting platelet activation via collagen is not involved. BrdU pulse-labelling showed no change in the proliferative potential of cells associated with capillaries after platelet depletion. Inhibition of platelet activation by acetylsalicylic acid abolished sprouting, but not splitting angiogenesis, paralleling the response to platelet depletion. We conclude that platelets differentially regulate mechanisms of angiogenesis in vivo, likely via COX signalling. Since endothelial proliferation is not impaired, we propose a link between COX1 and induction of endothelial migration.     

Evidence suggesting a stimulatory role for interleukin-10 in erythropoiesis in vitro

Interleukin-10 (IL-10) has been shown to exert anti-inflammatory effects by suppressing macrophage proliferation and inhibiting cytokine production. In this study we show that in the presence of erythropoietin (EPO), the addition of IL-10 results in a significant dose-dependent increase in both Burst Forming Unit-Erythroid (BFU-E) and Colony Forming Unit-Erythroid (CFU-E) colony growth in both serum-containing and serum-free murine cultures in vitro. IL-10 acts at the later stages of erythroid cell proliferation and differentiation as the increase in colony number was greater in CFU-E than in BFU-E, and was similar when IL-10 was added to BFU-E cultures at the time of culture initiation as when its addition to culture was delayed for 7 days. Furthermore, no increase in BFU-E colony number was noted when IL-10, added at the time of culture initiation, was neutralized by the addition to culture of a monoclonal anti-IL-10 antibody up to 7 days later. The increases in BFU-E by IL-10 addition were not the result of prolongation of BFU-E colony lifespan, which was not significantly different in IL-10 treated and control cultures, respectively. Rather IL-10 stimulated the proliferation of erythroid clusters that were now large enough to be recognized as colonies. IL-10-induced stimulation of erythropoiesis appeared to be independent of its inhibitory effects on macrophage function, as stimulation of erythroid colony growth was similar in macrophage-containing and depleted cultures. Studies to determine if the IL-10 effect was direct or indirect yielded equivocal results. A limiting dilution assay suggested a direct effect. However, a log/log dose response curve with IL-10 did not pass through the origin suggesting an indirect effect. These studies indicate that IL-10 acts synergistically with EPO to significantly increase stimulation of erythroid differentiation and proliferation in vitro and may be involved in the regulation of normal erythropoiesis in vivo. © 1996 Wiley-Liss, Inc.     

Molecular Evidence for High Frequency of Multiple Paternity in a Freshwater Shrimp Species Caridina ensifera

Background

Molecular genetic analyses of parentage provide insights into mating systems. Although there are 22,000 members in Malacostraca, not much has been known about mating systems in Malacostraca. The freshwater shrimp Caridina ensifera blue, is a new species belonging to Malacostraca which was discovered recently in Sulawesi, Indonesia. Due to its small body size and low fecundity, this species is an ideal species to study the occurrence and frequency of multiple paternity and to understand of how the low fecundity species persist and evolve.

Methodology/Principal Findings

In this study, we developed four polymorphic microsatellites from C. ensifera and applied them to investigate the occurrence and frequency of multiple paternity in 20 C. ensifera broods caught from Lake Matano, Sulawesi. By genotyping the mother and all offspring from each brood we discovered multiple paternity in all 20 broods. In most of the 20 broods, fathers contributed skewed numbers of offspring and there was an apparent inverse correlation between reproductive success of sires and their relatedness to mothers.

Conclusions/Significance

Our results in combination with recent reports on multiple paternity in crayfish, crab and lobster species suggests that multiple paternity is common in Malacostraca. Skewed contribution of fathers to the numbers of offspring and inverse correlation between reproductive success of sires and their relatedness to mothers suggest that sperm competition occurred and/or pre- and postcopulatory female choice happen, which may be important for avoiding the occurrence of inbreeding and optimize genetic variation in offspring and for persistence and evolution of low fecundity species.     

Sex in Cheese: Evidence for Sexuality in the Fungus Penicillium roqueforti

Although most eukaryotes reproduce sexually at some moment of their life cycle, as much as a fifth of fungal species were thought to reproduce exclusively asexually. Nevertheless, recent studies have revealed the occurrence of sex in some of these supposedly asexual species. For industrially relevant fungi, for which inoculums are produced by clonal-subcultures since decades, the potentiality for sex is of great interest for strain improvement strategies. Here, we investigated the sexual capability of the fungus Penicillium roqueforti, used as starter for blue cheese production. We present indirect evidence suggesting that recombination could be occurring in this species. The screening of a large sample of strains isolated from diverse substrates throughout the world revealed the existence of individuals of both mating types, even in the very same cheese. The MAT genes, involved in fungal sexual compatibility, appeared to evolve under purifying selection, suggesting that they are still functional. The examination of the recently sequenced genome of the FM 164 cheese strain enabled the identification of the most important genes known to be involved in meiosis, which were found to be highly conserved. Linkage disequilibria were not significant among three of the six marker pairs and 11 out of the 16 possible allelic combinations were found in the dataset. Finally, the detection of signatures of repeat induced point mutations (RIP) in repeated sequences and transposable elements reinforces the conclusion that P. roqueforti underwent more or less recent sex events. In this species of high industrial importance, the induction of a sexual cycle would open the possibility of generating new genotypes that would be extremely useful to diversify cheese products.     

Evidence for coloration plasticity in the yellow‐bellied toad,Bombina variegata

Phenotypic adaptation in terms of background color matching to the local habitat is an important mechanism for survival in prey species. Thus, intraspecific variation in cryptic coloration is expected among localities with dissimilar habitat features (e.g., soil, vegetation). Yellow‐bellied toads (Bombina variegata) display a dark dorsal coloration that varies between populations, assumed to convey crypsis. In this study, we explored I) geographic variation in dorsal coloration and II) coloration plasticity in B. variegata from three localities differing in substrate coloration. Using avian visual modeling, we found that the brightness contrasts of the cryptic dorsa were significantly lower on the local substrates than substrates of other localities. In experiments, individuals from one population were able to quickly change the dorsal coloration to match a lighter substrate. We conclude that the environment mediates an adaptation in cryptic dorsal coloration. We suggest further studies to test the mechanisms by which the color change occurs and explore the adaptive potential of coloration plasticity on substrates of varying brightness in B. variegata and other species.     

Suggestive Evidence for Darwinian Selection against Asparagine-Linked Glycans of Plasmodium falciparum and Toxoplasma gondii

We are interested in asparagine-linked glycans (N-glycans) of Plasmodium falciparum and Toxoplasma gondii, because their N-glycan structures have been controversial and because we hypothesize that there might be selection against N-glycans in nucleus-encoded proteins that must pass through the endoplasmic reticulum (ER) prior to threading into the apicoplast. In support of our hypothesis, we observed the following. First, in protists with apicoplasts, there is extensive secondary loss of Alg enzymes that make lipid-linked precursors to N-glycans. Theileria makes no N-glycans, and Plasmodium makes a severely truncated N-glycan precursor composed of one or two GlcNAc residues. Second, secreted proteins of Toxoplasma, which uses its own 10-sugar precursor (Glc₃Man₅GlcNAc₂) and the host 14-sugar precursor (Glc₃Man₉GlcNAc₂) to make N-glycans, have very few sites for N glycosylation, and there is additional selection against N-glycan sites in its apicoplast-targeted proteins. Third, while the GlcNAc-binding Griffonia simplicifolia lectin II labels ER, rhoptries, and surface of plasmodia, there is no apicoplast labeling. Similarly, the antiretroviral lectin cyanovirin-N, which binds to N-glycans of Toxoplasma, labels ER and rhoptries, but there is no apicoplast labeling. We conclude that possible selection against N-glycans in protists with apicoplasts occurs by eliminating N-glycans (Theileria), reducing their length (Plasmodium), or reducing the number of N-glycan sites (Toxoplasma). In addition, occupation of N-glycan sites is markedly reduced in apicoplast proteins versus some secretory proteins in both Plasmodium and Toxoplasma.Animals, fungi, and plants synthesize Asn-linked glycans (N-glycans) by means of a lipid-linked precursor containing 14 sugars (dolichol-PP-Glc₃Man₉GlcNAc₂) (26). Recently we used bioinformatics and experimental methods to show that numerous protists are missing sets of glycosyltransferases (Alg1 to Alg14) and so make truncated N-glycan precursors containing 0 to 11 sugars (46). For example, Entamoeba histolytica, which causes dysentery, makes N-glycan precursors that contain seven sugars (Man₅GlcNAc₂) (33). Giardia lamblia, a cause of diarrhea, makes N-glycan precursors that contain just GlcNAc₂ (41). N-glycan precursors may be identified by metabolic labeling with radiolabeled mannose (Entamoeba) or glucosamine (Giardia) (46). Unprocessed N-glycans of each protist may be recognized by wheat germ agglutinin 1 (WGA-1) (GlcNAc₂ of Giardia) or by the antiretroviral lectin cyanovirin-N (Man₅GlcNAc₂ of Entamoeba) (2, 33, 41).N-glycans are transferred from lipid-linked precursors to sequons (Asn-Xaa-Ser or Asn-Xaa-Thr, where Xaa cannot be Pro) on nascent peptides by an oligosaccharyltransferase (OST) (28). For the most part, transfer of N-glycans by the OST is during translocation, although there are human and Trypanosoma OSTs that transfer N-glycans after translocation (34, 45).N-glycan-dependent quality control (QC) systems for protein folding and endoplasmic reticulum (ER)-associated degradation (ERAD), which are present in most eukaryotes, are missing from Giardia and a few other protists that make truncated N-glycans (5, 26, 53). There is positive Darwinian selection for sequons (sites of N-glycans) that contain Thr in secreted and membrane proteins of organisms that have N-glycan-dependent QC (12). This selection occurs for the most part by an increased probability that Asn and Thr will be present in sequons rather than elsewhere in secreted and membrane proteins. In contrast, there is no selection on sequons that contain Ser, and there is no selection on sequons in the secreted proteins of organisms that lack N-glycan-dependent QC.For numerous reasons, we are interested in the N-glycans of Plasmodium falciparum and Toxoplasma gondii, which cause severe malaria and disseminated infections, respectively.(i) There has been controversy for a long time as to whether Plasmodium makes N-glycans. While some investigators identified a 14-sugar Plasmodium N-glycan resembling that of the human host (29), others identified no N-glycans (6, 22).(ii) There is also controversy concerning whether the N-glycans of Toxoplasma, after removal of Glc by glucosidases in the ER lumen, contain either 7 sugars (Man₅GlcNAc₂), like Entamoeba (32, 33), or 11 sugars (Man₉GlcNAc₂), like the human host (16, 19, 26). If it is Man₅GlcNAc₂, then Toxoplasma uses the dolichol-PP-linked glycan predicted by its set of Alg enzymes (32, 46). If it is Man₉GlcNAc₂, then Toxoplasma uses the dolichol-PP-linked glycan of the host cell (16, 19, 26).(iii) Both Plasmodium and Toxoplasma are missing proteins involved in N-glycan-dependent QC of protein folding (5).(iv) We hypothesize that there may be negative selection against N-glycans in Plasmodium and Toxoplasma, because the N-glycans added in the ER lumen during translocation will likely interfere with threading of nucleus-encoded apicoplast proteins into a nonphotosynthetic, chloroplast-derived organelle called the apicoplast (21, 35, 37, 48, 52, 54). Nucleus-encoded apicoplast proteins have a bipartite signal at the N terminus, which targets proteins first to the lumen of the ER and second to lumen of the apicoplast. This bipartite signal has been used in transformed plasmodia where green fluorescent protein (GFP) is targeted to the apicoplast with the bipartite signal of the acyl carrier protein (ACP_leader-GFP), to the secretory system with the signal sequence only (ACP_signal-GFP), and to the cytosol with the organelle-targeting transit peptide only (ACP_transit-GFP) (55). Similar constructs have been used to characterize signals that target nucleus-encoded proteins of Toxoplasma to the apicoplast (11, 25).Here we use a combination of bioinformatic, biochemical, and morphological methods to characterize the N-glycans of Plasmodium and Toxoplasma and to test our hypothesis that there is negative selection against N-glycans in protists with apicoplasts.