Protein targeting into complex diatom plastids: functional characterisation of a specific targeting motif

Plastids of diatoms and related algae evolved by secondary endocytobiosis, the uptake of a eukaryotic alga into a eukaryotic host cell and its subsequent reduction into an organelle. As a result diatom plastids are surrounded by four membranes. Protein targeting of nucleus encoded plastid proteins across these membranes depends on N-terminal bipartite presequences consisting of a signal and a transit peptide-like domain. Diatoms and cryptophytes share a conserved amino acid motif of unknown function at the cleavage site of the signal peptides (ASAFAP), which is particularly important for successful plastid targeting. Screening genomic databases we found that in rare cases the very conserved phenylalanine within the motif may be replaced by tryptophan, tyrosine or leucine. To test such unusual presequences for functionality and to better understand the role of the motif and putative receptor proteins involved in targeting, we constructed presequence:GFP fusion proteins with or without modifications of the “ASAFAP”-motif and expressed them in the diatom Phaeodactylum tricornutum. In this comprehensive mutational analysis we found that only the aromatic amino acids phenylalanine, tryptophan, tyrosine and the bulky amino acid leucine at the +1 position of the predicted signal peptidase cleavage site allow plastid import, as expected from the sequence comparison of native plastid targeting presequences of P. tricornutum and the cryptophyte Guillardia theta. Deletions within the signal peptide domains also impaired plastid import, showing that the presence of F at the N-terminus of the transit peptide together with a cleavable signal peptide is crucial for plastid import. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. A. Gruber and S. Vugrinec contributed equally to this work.     

Der1-mediated preprotein import into the periplastid compartment of chromalveolates?

Phototrophic chromalveolates possess plastids surrounded by either 3 or 4 membranes, revealing their secondary endosymbiotic origin from an engulfed eukaryotic alga. In cryptophytes, a member of the chromalveolates, the organelle is embedded within a designated region of the host's rough endoplasmic reticulum (RER). Its eukaryotic compartments other than the plastid were reduced to the mere remains of its former cytosol, the periplastid compartment (PPC, PP space), and its nucleus, the nucleomorph, separated from the RER by its former plasma membrane, the periplast membrane (PPM). In the nucleomorph genome of the cryptophyte Guillardia theta, we identified several genes sharing homology with components of the ER-associated degradation (ERAD) machinery of yeast and higher eukaryotes, namely ORF201 and ORF477, homologs of membrane-bound proteins, Der1p (Degradation in the ER protein 1) and the RING-finger ubiquitin ligase Hrd1, and a truncated version of Udf1, a cofactor of Cdc48, a lumenal ATPase. Exemplarily, studies on the Der1-homolog ORF201 showed that this protein partially rescued a yeast deletion mutant, indicating the existence of a functional PPC-specific ERAD-like system in cryptophytes. With the noninvestigated exception of haptophytes a phylogenetically and mechanistically related system is apparently present in all chromalveolates with 4 membrane-bound plastids because amongst others, PPC-specific Derlins (Der1-like proteins), CDC48 and its cofactor Ufd1 were identified in the nuclear genomes of diatoms and apicomplexa. These proteins are equipped with the required topogenic signals to direct them into the periplastid compartment of their secondary symbionts. Based on our findings, we suggest that all chromalveolates with 4 membrane-bound plastids express an ERAD-derived machinery in the PPM of their secondary plastid, coexisting physically and systematically adjacent to the host's own ERAD system. We propose herewith that this system was functionally adapted to mediate transport of nucleus-encoded PPC/plastid preproteins from the RER into the periplastid space.     

Translocation of a phycoerythrin alpha subunit across five biological membranes

Cryptophytes, unicellular algae, evolved by secondary endosymbiosis and contain plastids surrounded by four membranes. In contrast to cyanobacteria and red algae, their phycobiliproteins do not assemble into phycobilisomes and are located within the thylakoid lumen instead of the stroma. We identified two gene families encoding phycoerythrin alpha and light-harvesting complex proteins from an expressed sequence tag library of the cryptophyte Guillardia theta. The proteins bear a bipartite topogenic signal responsible for the transport of nuclear encoded proteins via the ER into the plastid. Analysis of the phycoerythrin alpha sequences revealed that more than half of them carry an additional, third topogenic signal comprising a twin arginine motif, which is indicative of Tat (twin arginine transport)-specific targeting signals. We performed import studies with several derivatives of one member using a diatom transformation system, as well as intact chloroplasts and thylakoid vesicles isolated from pea. We demonstrated the different targeting properties of each individual part of the tripartite leader and show that phycoerythrin alpha is transported across the thylakoid membrane into the thylakoid lumen and protease-protected. Furthermore, we showed that thylakoid transport of phycoerythrin alpha takes place by the Tat pathway even if the 36 amino acid long bipartite topogenic signal precedes the actual twin arginine signal. This is the first experimental evidence of a protein being targeted across five biological membranes.     

A novel family of single VWC-domain proteins in invertebrates

Using a combination of sequence analysis tools, a novel family of 13 short Drosophila melanogaster proteins with similarity to a single von Willebrand factor C-domain (SVC proteins) has been identified. SVCs are predicted to be secreted and a structural model for the family is proposed, using a known von Willebrand factor C-domain (VWC) structure as template. All SVCs possess eight cysteines, consistent with the loss of one bonded pair from the 10-cysteine canonical pattern of most VWC domains. Unlike most Drosophila genes, many SVCs are not expressed during development, and misexpression has no apparent effect on development or viability. SVCs appear to be restricted to arthropods. A role for SVCs in response to environmental challenges is proposed.     

Inhibitory effects of N-ethylmaleimide on insulin- and oxidant-stimulated sugar transport and on 125I-labelled insulin binding by rat soleus muscle

These experiments examined the effects of N-ethylmaleimide on insulin- and oxidant-stimulated sugar transport in soleus muscle in terms of the Thiol-Redox model for insulin-stimulated adipocyte sugar transport (Czech, M.P. (1976) J. Cell. Physiol. 89, 661-668). Brief exposure (1 min) to N-ethylmaleimide (0.3-10 mM) inhibited the stimulatory effect of insulin (0.1 U/ml) on D-[U-14C]xylose uptake by rat soleus muscle. N-Ethylmaleimide also inhibited the stimulatory effects of H2O2 (5 mM), diamide (0.2 mM) and vitamin K-5 (0.05 mM). This effect of N-ethylmaleimide on insulin action was paralleled by the inhibition of 125I-labelled insulin binding by the muscle. N-ethylmaleimide lowered muscle ATP; however, its effects on sugar transport and 125I-labelled insulin binding could be dissociated from its effect on ATP. Exposing muscles to insulin prior to N-ethylmaleimide did not abolish the inhibitory effect of sulphydryl blockade on insulin-stimulated sugar transport, but did reduce the effect of the inhibitor by 20-30%. Conversely, when muscles were first allowed to bind 125I-labelled insulin and then exposed to the inhibitor, there was no effect of N-ethylmaleimide on pre-bound insulin. Exposure to diamide or vitamin K-5 before N-ethylmaleimide (1 mM) attenuated the inhibitory effect of sulphydryl blockade but no protective effect was observed with H2O2. None of the oxidants protected against the inhibitory effect of 3 mM N-ethylmaleimide. It is concluded that there are two N-ethylmaleimide-sensitive sites involved in the activation of muscle sugar transport at the post-receptor level. One of these would appear to be similar to the Thiol-Redox site described in the adipocyte; the other site appears to be an essential sulphydryl group whose function does not involve oxidation to a disulphide.     

N-Terminal Presequence-Independent Import of Phosphofructokinase into Hydrogenosomes of Trichomonas vaginalis

Mitochondrial evolution entailed the origin of protein import machinery that allows nuclear-encoded proteins to be targeted to the organelle, as well as the origin of cleavable N-terminal targeting sequences (NTS) that allow efficient sorting and import of matrix proteins. In hydrogenosomes and mitosomes, reduced forms of mitochondria with reduced proteomes, NTS-independent targeting of matrix proteins is known. Here, we studied the cellular localization of two glycolytic enzymes in the anaerobic pathogen Trichomonas vaginalis: PP_i-dependent phosphofructokinase (TvPP_i-PFK), which is the main glycolytic PFK activity of the protist, and ATP-dependent PFK (TvATP-PFK), the function of which is less clear. TvPP_i-PFK was detected predominantly in the cytosol, as expected, while all four TvATP-PFK paralogues were imported into T. vaginalis hydrogenosomes, although none of them possesses an NTS. The heterologous expression of TvATP-PFK in Saccharomyces cerevisiae revealed an intrinsic capability of the protein to be recognized and imported into yeast mitochondria, whereas yeast ATP-PFK resides in the cytosol. TvATP-PFK consists of only a catalytic domain, similarly to “short” bacterial enzymes, while ScATP-PFK includes an N-terminal extension, a catalytic domain, and a C-terminal regulatory domain. Expression of the catalytic domain of ScATP-PFK and short Escherichia coli ATP-PFK in T. vaginalis resulted in their partial delivery to hydrogenosomes. These results indicate that TvATP-PFK and the homologous ATP-PFKs possess internal structural targeting information that is recognized by the hydrogenosomal import machinery. From an evolutionary perspective, the predisposition of ancient ATP-PFK to be recognized and imported into hydrogenosomes might be a relict from the early phases of organelle evolution.     

BIOSYSTEMATIC STUDIES IN THE BOUTELOUA CURTIPENDULA COMPLEX. I. THE ANEUPLOID RHIZOMATOUS B. CURTIPENDULA OF TEXAS

Gould , F. W., and Z. J. Kapadia . (A. & M. College of Texas, College Station.) Biosystematic studies in the Bouteloua curtipendula complex. I. The aneuploid rhizomatous B. curtipendula of Texas. Amer. Jour. Bot. 49(8): 887–891. Illus. 1962.—Widespread throughout central U.S. is a rhizomatous form of B. curtipendula that basically is tetraploid (2n = 40). In the southwest the predominant type is a caespitose aneuploid with a high chromosome number (2n = ca. 80 to 2n = ca. 102). The present study has shown the presence of an extensive series of rhizomatous aneuploids in central Texas, with chromosome numbers ranging from 2n = 41 to 2n = 64. The distribution of these plants is centered about the region of overlap in the ranges of the 2 previously mentioned types. Available evidence indicates that the rhizomatous aneuploids have arisen through hybridization of the caespitose aneuploids and the rhizomatous tetraploids.     

Male-female affiliative relationships in naturally occurring ringtailed lemurs (Lemur catta) at the Beza-Mahafaly Reserve,Madagascar

Affiliative behavior between adult male and female ringtailed lemurs was examined as part of a project concerning male affiliation with conspecifics of all age/sex classes. Males in three social groups were studied over a 12 month period. Male-female preferred partnerships existed, and were variable according to reproductive season. Dominance rank, age, or tenure of the male did not appear to affect either the number of partnerships or frequency of affiliative behaviors that males had with females. However, males residing in groups with fewer males exhibited both higher frequencies of affiliative interactions with females and were nearest neighbors to females more often than males living in a group containing more males. Females were found to be responsible for proximity maintenance of male-female dyads in the majority of cases. Neither reproductive season nor seasonal availability of food resources strongly affected the frequency of affiliative interactions between males and females, it is proposed that an important aspect of successful group membership for male ringtailed lemurs relates to the development of social relationships with adult females. Males can benefit from such relationships in terms of greater centrality to the spatial core of the group, which can result in enhanced predator protection, greater opportunities for social contact, and potentially greater access to estrous females. © 1996 Wiley-Liss, Inc.     

Hexagonal shaped ice spicules in frozen antifreeze protein solutions

In the presence of antifreeze proteins from both Antarctic and Arctic fishes, water freezes in the form of long c-axis spikes or spicular-like crystals. Transmission electron microscopy of the Pt/C replicas of the freeze fractured spicular ice in a small capillary revealed the presence of many hexagonally shaped structures whose cross-sectional dimensions were between 0.5 and 10 microm. Well-defined parallel faces were associated with most fractured and etched spicules. When fracture planes occurred near the tip of a spicule, well-defined pyramidal faces were apparent. Steps were sometimes associated with these pyramidal spicular crystal faces. On some of the replicas obvious roughening of certain crystal faces of the spicule was observed, suggesting that the antifreeze proteins may have adsorbed to those faces.