Essential Import Research Articles

Biochemical and neurophysiological effects of deficiency of the mitochondrial import protein TIMM50.

TIMM50, an essential TIM23 complex subunit, is suggested to facilitate the import of ∼60% of the mitochondrial proteome. In this study, we characterized a TIMM50 disease causing mutation in human fibroblasts and noted significant decreases in TIM23 core protein levels (TIMM50, TIMM17A/B, and TIMM23). Strikingly, TIMM50 deficiency had no impact on the steady state levels of most of its putative substrates, suggesting that even low levels of a functional TIM23 complex are sufficient to maintain the majority of TIM23 complex-dependent mitochondrial proteome. As TIMM50 mutations have been linked to severe neurological phenotypes, we aimed to characterize TIMM50 defects in manipulated mammalian neurons. TIMM50 knockdown in mouse neurons had a minor effect on the steady state level of most of the mitochondrial proteome, supporting the results observed in patient fibroblasts. Amongst the few affected TIM23 substrates, a decrease in the steady state level of components of the intricate oxidative phosphorylation and mitochondrial ribosome complexes was evident. This led to declined respiration rates in fibroblasts and neurons, reduced cellular ATP levels and defective mitochondrial trafficking in neuronal processes, possibly contributing to the developmental defects observed in patients with TIMM50 disease. Finally, increased electrical activity was observed in TIMM50 deficient mice neuronal cells, which correlated with reduced levels of KCNJ10 and KCNA2 plasma membrane potassium channels, likely underlying the patients' epileptic phenotype.

CALHM2 is a mitochondrial protein import channel that regulates fatty acid metabolism.

For mitochondrial metabolism to occur in the matrix, multiple proteins must be imported across the two (inner and outer) mitochondrial membranes. Classically, two protein import channels, TIM/TOM, are known to perform this function, but whether other protein import channels exist is not known. Here, using super-resolution microscopy, proteomics, and electrophysiological techniques, we identify CALHM2 as the import channel for the ECHA subunit of the mitochondrial trifunctional protein (mTFP), which catalyzes β-oxidation of fatty acids in the mitochondrial matrix. We find that CALHM2 sits specifically at the inner mitochondrial and cristae membranes and is critical for membrane morphology. Depletion of CALHM2 leads to a mislocalization of ECHA outside of the mitochondria leading to severe cellular metabolic defects. These defects include cytosolic accumulation of fatty acids, depletion of tricarboxylic acid cycle enzymes and intermediates, and reduced cellular respiration. Our data identify CALHM2 as an essential protein import channel that is critical for fatty acid- and glucose-dependent aerobic metabolism.

Yeast 26S proteasome nuclear import is coupled to nucleus-specific degradation of the karyopherin adaptor protein Sts1

In eukaryotes, the ubiquitin–proteasome system is an essential pathway for protein degradation and cellular homeostasis. 26S proteasomes concentrate in the nucleus of budding yeast Saccharomyces cerevisiae due to the essential import adaptor protein Sts1 and the karyopherin-α protein Srp1. Here, we show that Sts1 facilitates proteasome nuclear import by recruiting proteasomes to the karyopherin-α/β heterodimer. Following nuclear transport, the karyopherin proteins are likely separated from Sts1 through interaction with RanGTP in the nucleus. RanGTP-induced release of Sts1 from the karyopherin proteins initiates Sts1 proteasomal degradation in vitro. Sts1 undergoes karyopherin-mediated nuclear import in the absence of proteasome interaction, but Sts1 degradation in vivo is only observed when proteasomes successfully localize to the nucleus. Sts1 appears to function as a proteasome import factor during exponential growth only, as it is not found in proteasome storage granules (PSGs) during prolonged glucose starvation, nor does it appear to contribute to the rapid nuclear reimport of proteasomes following glucose refeeding and PSG dissipation. We propose that Sts1 acts as a single-turnover proteasome nuclear import factor by recruiting karyopherins for transport and undergoing subsequent RanGTP-initiated ubiquitin-independent proteasomal degradation in the nucleus.

SUMOylation contributes to proteostasis of the chloroplast protein import receptor TOC159 during early development.

Chloroplast biogenesis describes the transition of non-photosynthetic proplastids to photosynthetically active chloroplasts in the cells of germinating seeds. Chloroplast biogenesis requires the import of thousands of nuclear-encoded preproteins by essential import receptor TOC159. We demonstrate that the small ubiquitin-related modifier (SUMO) pathway crosstalks with the ubiquitin-proteasome pathway to affect TOC159 stability during early plant development. We identified a SUMO3-interacting motif (SIM) in the TOC159 GTPase domain and a SUMO3 covalent SUMOylation site in the membrane domain. A single K to R substitution (K1370R) in the M-domain disables SUMOylation. Compared to wild-type TOC159, TOC159K1370R was destabilized under UPS-inducing stress conditions. However, TOC159K1370R recovered to same protein level as wild-type TOC159 in the presence of a proteasome inhibitor. Thus, SUMOylation partially stabilizes TOC159 against UPS-dependent degradation under stress conditions. Our data contribute to the evolving model of tightly controlled proteostasis of the TOC159 import receptor during proplastid to chloroplast transition.

Coexpressed subunits of dual genetic origin define a conserved supercomplex mediating essential protein import into chloroplasts

In photosynthetic eukaryotes, thousands of proteins are translated in the cytosol and imported into the chloroplast through the concerted action of two translocons-termed TOC and TIC-located in the outer and inner membranes of the chloroplast envelope, respectively. The degree to which the molecular composition of the TOC and TIC complexes is conserved over phylogenetic distances has remained controversial. Here, we combine transcriptomic, biochemical, and genetic tools in the green alga Chlamydomonas (Chlamydomonas reinhardtii) to demonstrate that, despite a lack of evident sequence conservation for some of its components, the algal TIC complex mirrors the molecular composition of a TIC complex from Arabidopsis thaliana. The Chlamydomonas TIC complex contains three nuclear-encoded subunits, Tic20, Tic56, and Tic100, and one chloroplast-encoded subunit, Tic214, and interacts with the TOC complex, as well as with several uncharacterized proteins to form a stable supercomplex (TIC-TOC), indicating that protein import across both envelope membranes is mechanistically coupled. Expression of the nuclear and chloroplast genes encoding both known and uncharacterized TIC-TOC components is highly coordinated, suggesting that a mechanism for regulating its biogenesis across compartmental boundaries must exist. Conditional repression of Tic214, the only chloroplast-encoded subunit in the TIC-TOC complex, impairs the import of chloroplast proteins with essential roles in chloroplast ribosome biogenesis and protein folding and induces a pleiotropic stress response, including several proteins involved in the chloroplast unfolded protein response. These findings underscore the functional importance of the TIC-TOC supercomplex in maintaining chloroplast proteostasis.

SUMOylation contributes to proteostasis of the chloroplast protein import receptor TOC159 during early development

Chloroplast biogenesis describes the transition of non-photosynthetic proplastids to photosynthetically active chloroplasts in the cells of germinating seeds. Chloroplast biogenesis requires the import of thousands of nuclear-encoded preproteins by essential import receptor TOC159. We demonstrate that the small ubiquitin-related modifier (SUMO) pathway crosstalks with the ubiquitin–proteasome pathway to affect TOC159 stability during early plant development. We identified a SUMO3-interacting motif (SIM) in the TOC159 GTPase domain and a SUMO3 covalent SUMOylation site in the membrane domain. A single K to R substitution (K1370R) in the M-domain disables SUMOylation. Compared to wild-type TOC159, TOC159K1370R was destabilized under UPS-inducing stress conditions. However, TOC159K1370R recovered to same protein level as wild-type TOC159 in the presence of a proteasome inhibitor. Thus, SUMOylation partially stabilizes TOC159 against UPS-dependent degradation under stress conditions. Our data contribute to the evolving model of tightly controlled proteostasis of the TOC159 import receptor during proplastid to chloroplast transition.

CHCHD4 regulates tumour proliferation and EMT-related phenotypes, through respiratory chain-mediated metabolism

BackgroundMitochondrial oxidative phosphorylation (OXPHOS) via the respiratory chain is required for the maintenance of tumour cell proliferation and regulation of epithelial to mesenchymal transition (EMT)-related phenotypes through mechanisms that are not fully understood. The essential mitochondrial import protein coiled-coil helix coiled-coil helix domain-containing protein 4 (CHCHD4) controls respiratory chain complex activity and oxygen consumption, and regulates the growth of tumours in vivo. In this study, we interrogate the importance of CHCHD4-regulated mitochondrial metabolism for tumour cell proliferation and EMT-related phenotypes, and elucidate key pathways involved.ResultsUsing in silico analyses of 967 tumour cell lines, and tumours from different cancer patient cohorts, we show that CHCHD4 expression positively correlates with OXPHOS and proliferative pathways including the mTORC1 signalling pathway. We show that CHCHD4 expression significantly correlates with the doubling time of a range of tumour cell lines, and that CHCHD4-mediated tumour cell growth and mTORC1 signalling is coupled to respiratory chain complex I (CI) activity. Using global metabolomics analysis, we show that CHCHD4 regulates amino acid metabolism, and that CHCHD4-mediated tumour cell growth is dependent on glutamine. We show that CHCHD4-mediated tumour cell growth is linked to CI-regulated mTORC1 signalling and amino acid metabolism. Finally, we show that CHCHD4 expression in tumours is inversely correlated with EMT-related gene expression, and that increased CHCHD4 expression in tumour cells modulates EMT-related phenotypes.ConclusionsCHCHD4 drives tumour cell growth and activates mTORC1 signalling through its control of respiratory chain mediated metabolism and complex I biology, and also regulates EMT-related phenotypes of tumour cells.

Newly born peroxisomes are a hybrid of mitochondrial and ER-derived pre-peroxisomes.

Peroxisomes function together with mitochondria in a number of essential biochemical pathways, from bile acid synthesis to fatty acid oxidation. Peroxisomes grow and divide from pre-existing organelles, but can also emerge de novo in the cell. The physiological regulation of de novo peroxisome biogenesis remains unclear, and it is thought that peroxisomes emerge from the endoplasmic reticulum in both mammalian and yeast cells. However, in contrast to the yeast system, a number of integral peroxisomal membrane proteins are imported into mitochondria in mammalian cells in the absence of peroxisomes, including Pex3, Pex12, Pex13, Pex14, Pex26, PMP34 and ALDP. Overall, the mitochondrial localization of peroxisomal membrane proteins in mammalian cells has largely been considered a mis-targeting artefact in which de novo biogenesis occurs exclusively from endoplasmic reticulum-targeted peroxins. Here, in following the generation of new peroxisomes within human patient fibroblasts lacking peroxisomes, we show that the essential import receptors Pex3 and Pex14 target mitochondria, where they are selectively released into vesicular pre-peroxisomal structures. Maturation of pre-peroxisomes containing Pex3 and Pex14 requires fusion with endoplasmic reticulum-derived vesicles carrying Pex16, thereby providing full import competence. These findings demonstrate the hybrid nature of newly born peroxisomes, expanding their functional links to mitochondria.

Evidence for the Role of BAG3 in Mitochondrial Quality Control in Cardiomyocytes.

Mitochondrial abnormalities impact the development of myofibrillar myopathies. Therefore, understanding the mechanisms underlying the removal of dysfunctional mitochondria from cells is of great importance toward understanding the molecular events involved in the genesis of cardiomyopathy. Earlier studies have ascribed a role for BAG3 in the development of cardiomyopathy in experimental animals leading to the identification of BAG3 mutations in patients with heart failure which may play a part in the onset of disease development and progression. BAG3 is co-chaperone of heat shock protein 70 (HSP70), which has been shown to modulate apoptosis and autophagy, in several cell models. In this study, we explore the potential role of BAG3 in mitochondrial quality control. We demonstrate that siRNA mediated suppression of BAG3 production in neonatal rat ventricular cardiomyocytes (NRVCs) significantly elevates the level of Parkin, a key component of mitophagy. We found that both BAG3 and Parkin are recruited to depolarized mitochondria and promote mitophagy. Suppression of BAG3 in NRVCs significantly reduces autophagy flux and eliminates clearance of Tom20, an essential import receptor for mitochondria proteins, after induction of mitophagy. These observations suggest that BAG3 is critical for the maintenance of mitochondrial homeostasis under stress conditions, and disruptions in BAG3 expression impact cardiomyocyte function. J. Cell. Physiol. 232: 797-805, 2017. © 2016 Wiley Periodicals, Inc.

Crystal structure of the karyopherin Kap121p bound to the extreme C-terminus of the protein phosphatase Cdc14p

In Saccharomyces cerevisiae, the protein phosphatase Cdc14p is an antagonist of mitotic cyclin-dependent kinases and is a key regulator of late mitotic events such as chromosome segregation, spindle disassembly and cytokinesis. The activity of Cdc14p is controlled by cell-cycle dependent changes in its association with its competitive inhibitor Net1p (also known as Cfi1p) in the nucleolus. For most of the cell cycle up to metaphase, Cdc14p is sequestered in the nucleolus in an inactive state. During anaphase, Cdc14p is released from Net1p, spreads into the nucleus and cytoplasm, and dephosphorylates key mitotic targets. Although regulated nucleocytoplasmic shuttling of Cdc14p has been suggested to be important for exit from mitosis, the mechanism underlying Cdc14p nuclear trafficking remains poorly understood. Here we show that the C-terminal region (residues 517–551) of Cdc14p can function as a nuclear localization signal (NLS) in vivo and also binds to Kap121p (also known as Pse1p), an essential nuclear import carrier in yeast, in a Gsp1p-GTP-dependent manner in vitro. Moreover we report a crystal structure, at 2.4 Å resolution, of Kap121p bound to the C-terminal region of Cdc14p. The structure and structure-based mutational analyses suggest that either the last five residues at the extreme C-terminus of Cdc14p (residues 547–551; Gly-Ser-Ile-Lys-Lys) or adjacent residues with similar sequence (residues 540–544; Gly-Gly-Ile-Arg-Lys) can bind to the NLS-binding site of Kap121p, with two residues (Ile in the middle and Lys at the end of the five residues) of Cdc14p making key contributions to the binding specificity. Based on comparison with other structures of Kap121p-ligand complexes, we propose “IK-NLS” as an appropriate term to refer to the Kap121p-specific NLS.

Jungle Fever: Exploring Madness and Medicine in Twentieth Century Tropical Narratives by Charlotte Rogers (review)

ROGERS, CHARLOTTE. Jungle Fever: Exploring Madness and Medicine in Twentieth Century Tropical Narratives. Nashville: Vanderbilt UP, 2012. ix + 234 pp.Environmental determinism arose in Latin America particularly during the late nineteenth and early twentieth centuries when positivism was in vogue and when Latin American nations thirsted for their own exceptional claims to rooted identities in native soils. Regionalism's deep ties to place, to geographical and climatic specificity, became the paradoxically modem claim to identity and nationhood. tropical regions inspired particular fascination because of their radical departure from the environmental norms of European and imperial points of reference. They were the spaces where extravagant and violent nature broke the will of colonialism and where the regionalist novelist could offer diagnosis and, perhaps by implication, a cure for national ills.Charlotte Rogers's excellent study of tropical narratives is not exclusively focused on Latin America, but its essential import remains within the sphere of Latin American Studies, and more specifically within this particular modern turn toward the land. Its originality lies in its comparativism, drawing on readings of Joseph Conrad's Heart of Darkness and Andre Malraux's Way of the Kings that are offered as context for understanding the rise and function of discourses about medicine and madness in Latin American novelas de la selva. In addition to individual chapters devoted to these two novels, she also devotes chapters to Jose Eustasio Rivera's Vortex, Romulo Gallegos's Canaima, and Alejo Carpentier's Lost Steps. In these chapters and in her introduction and conclusion, however, she remains widely comparative, including brief but often insightful readings of such works as Virginia Woolf's Voyage Out, the stories of Horacio Quiroga and Jorge Luis Borges, Mario Vargas Llosa's Storyteller, and W. H. Hudson's Green Mansions. This is a rich and fruitful comparative context, and her readings are consistently well grounded bibliographically and linguistically, offering at times new and insightful understandings of this subgenre of literature that is so important to the history of Latin American literature.Her argument is that the determinism with which the regional turn began begins to break down particularly as a result of an increased interest in and awareness of mental illness. In other words, where it may have once been believed that the jungle was a geographical space of madness where peoples and cultures could not thrive as they had proved to in the more temperate climates of Europe, for example, this tropically induced mental breakdown begins to be diagnosed as an interior and more transverse form of madness. She explains, The descent into madness experienced by these characters indicates their twentieth-century creators' disenchantment with narratives of imperial expansion and their ostensible rationality. A deep undercurrent of pessimism runs beneath the surface of these adventure novels. They go far beyond a critique of colonialism: the fates of their protagonists undermine the Cartesian tenets of modern society and question the notion of progress upon which it is founded (1). In other words, the madness lies within.I think Rogers's book is important for several reasons. First, it exemplifies the benefits of more broadly comparative studies of Latin American literature. Rather than eroding the claims to Latin American specificity which some erroneously believe is the inevitable outcome of comparative work, the book manages to highlight the specific contributions these novels make to the discourse on Latin America, while also emphasizing their broader literary genealogy and their ambitions to underscore facets of the universal human condition-in this case, a condition of fundamental weakness and instability that should be a cause for more cross-cultural collaboration and solidarity. …

Malaria situation in Bulgaria and surveillance measures (1991-2000)

The malaria situation in Bulgaria during the past decade of the century was analysed and the evaluation of the risk of re-emerging of the diseases and factors determining it in the phase of maintanence of malaria eradication was carried out. The epidemiological analysis of malaria cases notified during the period 1991-2000 in Bulgaria is presented. Of a total of 382 malaria episodes registered, 363 (95.03%), were imported into Bulgaria from endemic countries, 1 (0.26%) occurred after haemotransfusion and 18 (4.71%) were indigenous introduced ones. Plasmodium falciparum accounted for 68.59% of all imported cases, P.vivax, for 23.82%, P.ovale, for 2.36%, P.malariae, for 1.85%. In 1.83% more than one species was detected, and in 6 cases, the species was not identified. The import of malaria came mainly from Africa-299 (82.37%) cases, followed by Asia-63 (17.36%) and Oceania-1 (0.27%). Four cases with falciparum malaria resulted in death. A great number of imported cases-231 (63.81%) was diagnosed during the potential malaria season in Bulgaria (April-October). The analysis of present entomological situation showed a high density of Anopheles population. As an indication for existence of potential risk of spread of indigenous malaria, especially in cases of delayed diagnosis and treatment, 18 indigenous cases of vivax malaria were recorded in 1995-1996 in the region of the town of Sandanski, for the first time after malaria eradication in 1965. The cases were classified as introduced ones. The analysis of the essential malaria import in Bulgaria, climatic conditions and presence of Anopheline species revealed a fairly high level of malariogenic potential. The stratification of the country was carried out in accordance with the degree of hazard of malaria spreading, and the territory was divided into three categories of regions: with high-, medium- and low-risk. The present situation, that probably will be valid for the future, too, requires a scientific based prognosis and improvement of a system of surveillance activities aiming at prevention of indigenous endemic and epidemic spreading of malaria in the country, as well as elaboration of adequate activities of the health network in case of reappearance of autochthonous cases.

The NIMH-CATIE Schizophrenia Study: What Did We Learn?

It has been over 10 years since the initiation of the National Institute of Mental Health (NIMH) Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE) study and 5 years since the first publication of its primary results (1). In this period, the initial report has been cited in the literature over 1,600 times (2) while more than 80 articles from the study’s extensive database (Table 1), as well as a book serving as an archive of the study’s results and implications (3), have been published. In the meantime, several more randomized trials comparing the effectiveness of antipsychotics have been completed (4–6), meta-analyses that bear on the findings of the CATIE study have been performed (7, 8), and commentaries on CATIE’s findings and critiques of its methodology have been published (9–11). All of these help us to view the CATIE study in a broader context and enable us to determine what we really learned from it. When the CATIE study was designed in 1999– 2000, the prevailing opinion of researchers and clinicians alike was that the newer (second-generation) antipsychotic drugs were vastly superior to the older (first-generation) antipsychotic drugs in efficacy and safety. This largely reflected the results of studies sponsored by the manufacturers of the new drugs (12, 13), marketing messages of pharmaceutical companies and the hopes of many who wanted better treatments. Indeed, the hypothesis and expectation of the CATIE study investigators was that the firstgeneration antipsychotic perphenazine would be inferior to the newer agents. Consequently, the finding that perphenazine was similar in effectiveness to most other medications had a profound effect that extended beyond the scientific and psychiatric communities to the lay public and various stakeholder groups. Somewhat sensational news reports decried the preferential use and greater cost of the newer medications and the marketing practices that led to them. For example, the September 21, 2005, editorial page of The New York Times opined, “A government-financed study has provided the strongest evidence yet that the system for approving and promoting drugs is badly out of whack. The study compared five drugs used to treat schizophrenia and found that most of the newest, most heavily prescribed drugs were no better than an older drug that is far cheaper. The nation is wasting billions of dollars on heavily marketed drugs that have never proved themselves in head-to-head competition against cheaper competitors” (14). But what did we really learn from the CATIE study? In this commentary, we summarize its major implications and their relevance to clinical practice. We will also address some of the study’s most relevant critiques.

A Systematic Screen to Discover and Analyze Apicoplast Proteins Identifies a Conserved and Essential Protein Import Factor

Parasites of the phylum Apicomplexa cause diseases that impact global health and economy. These unicellular eukaryotes possess a relict plastid, the apicoplast, which is an essential organelle and a validated drug target. However, much of its biology remains poorly understood, in particular its elaborate compartmentalization: four membranes defining four different spaces. Only a small number of organellar proteins have been identified in particular few proteins are known for non-luminal apicoplast compartments. We hypothesized that enlarging the catalogue of apicoplast proteins will contribute toward identifying new organellar functions and expand the realm of targets beyond a limited set of characterized pathways. We developed a bioinformatic screen based on mRNA abundance over the cell cycle and on phyletic distribution. We experimentally assessed 57 genes, and of 30 successful epitope tagged candidates eleven novel apicoplast proteins were identified. Of those, seven appear to target to the lumen of the organelle, and four localize to peripheral compartments. To address their function we then developed a robust system for the construction of conditional mutants via a promoter replacement strategy. We confirm the feasibility of this system by establishing conditional mutants for two selected genes – a luminal and a peripheral apicoplast protein. The latter is particularly intriguing as it encodes a hypothetical protein that is conserved in and unique to Apicomplexan parasites and other related organisms that maintain a red algal endosymbiont. Our studies suggest that this peripheral plastid protein, PPP1, is likely localized to the periplastid compartment. Conditional disruption of PPP1 demonstrated that it is essential for parasite survival. Phenotypic analysis of this mutant is consistent with a role of the PPP1 protein in apicoplast biogenesis, specifically in import of nuclear-encoded proteins into the organelle.

Import Oligomers Induce Positive Feedback to Promote Peroxisome Differentiation and Control Organelle Abundance

A fundamental question in cell biology is how cells control organelle composition and abundance. Woronin bodies are fungal peroxisomes centered on a crystalline core of the self-assembled HEX protein. Despite using the canonical peroxisome import machinery for biogenesis, Woronin bodies are scarce compared to the overall peroxisome population. Here, we show that HEX oligomers promote the differentiation of a subpopulation of peroxisomes, which become enlarged and highly active in matrix protein import. HEX physically associates with the essential matrix import peroxin, PEX26, and promotes its enrichment in the membrane of differentiated peroxisomes. In addition, a PEX26 mutant that disrupts differentiation produces increased numbers of aberrantly small Woronin bodies. Our data suggest a mechanism where HEX oligomers recruit a key component of the import machinery, which promotes the import of additional HEX. This type of positive feedback provides a basic mechanism for the production of an organelle subpopulation of distinct composition and abundance.

Proteomic alterations of distinct mitochondrial subpopulations in the type 1 diabetic heart: contribution of protein import dysfunction

Diabetic cardiomyopathy is associated with increased risk of heart failure in type 1 diabetic patients. Mitochondrial dysfunction is suggested as an underlying contributor to diabetic cardiomyopathy. Cardiac mitochondria are characterized by subcellular spatial locale, including mitochondria located beneath the sarcolemma, subsarcolemmal mitochondria (SSM), and mitochondria situated between the myofibrils, interfibrillar mitochondria (IFM). The goal of this study was to determine whether type 1 diabetic insult in the heart influences proteomic make-up of spatially distinct mitochondrial subpopulations and to evaluate the role of nuclear encoded mitochondrial protein import. Utilizing multiple proteomic approaches (iTRAQ and two-dimensional-differential in-gel electrophoresis), IFM proteomic make-up was impacted by type 1 diabetes mellitus to a greater extent than SSM, as evidenced by decreased abundance of fatty acid oxidation and electron transport chain proteins. Mitochondrial phosphate carrier and adenine nucleotide translocator, as well as inner membrane translocases, were decreased in the diabetic IFM (P < 0.05 for both). Mitofilin, a protein involved in cristae morphology, was diminished in the diabetic IFM (P < 0.05). Posttranslational modifications, including oxidations and deamidations, were most prevalent in the diabetic IFM. Mitochondrial heat shock protein 70 (mtHsp70) was significantly decreased in diabetic IFM (P < 0.05). Mitochondrial protein import was decreased in the diabetic IFM with no change in the diabetic SSM (P < 0.05). Taken together, these results indicate that mitochondrial proteomic alterations in the type 1 diabetic heart are more pronounced in the IFM. Further, proteomic alterations are associated with nuclear encoded mitochondrial protein import dysfunction and loss of an essential mitochondrial protein import constituent, mtHsp70, implicating this process in the pathogenesis of the diabetic heart.

Importin α is an essential nuclear import carrier adaptor required for proper sexual and asexual development and secondary metabolism in Aspergillus nidulans

In eukaryotes, the principal nuclear import pathway is driven by the importin α/β1 heterodimer. KapA, the Aspergillus nidulans importin α, is an essential protein. We generated a conditional allele, kapA31, mimicking the srp1-31 allele in Saccharomyces cerevisiae. KapA31 carries a Ser111Phe amino acid substitution which, at the restrictive temperature of 42 °C, reduces nuclear import of cargos containing classical nuclear-localization-sequences, cNLS. Using kapA31, we have demonstrated the role of the importin α in the nuclear accumulation of the light-dependent developmental regulator VeA. KapA have additional tasks in the cell, as reported for other members of the importin α family. KapA participates at different regulatory stages of asexual and sexual development, being required for the completion of both reproductive cycles with the formation of conidiospores and ascospores, respectively. Finally, KapA also mediates in different pathways of secondary metabolism having a dual role: positively for penicillin production and negatively for mycotoxin biosynthesis.

The Anchor-Away Technique: Rapid, Conditional Establishment of Yeast Mutant Phenotypes

The anchor-away (AA) technique depletes the nucleus of Saccharomyces cerevisiae of a protein of interest (the target) by conditional tethering to an abundant cytoplasmic protein (the anchor) by appropriate gene tagging and rapamycin-dependent heterodimerization. Taking advantage of the massive flow of ribosomal proteins through the nucleus during maturation, a protein of the large subunit was chosen as the anchor. Addition of rapamycin, due to formation of the ternary complex, composed of the anchor, rapamycin, and the target, then results in the rapid depletion of the target from the nucleus. All 43 tested genes displayed on rapamycin plates the expected defective growth phenotype. In addition, when examined functionally, specific mutant phenotypes were obtained within minutes. These are genes involved in protein import, RNA export, transcription, sister chromatid cohesion, and gene silencing. The AA technique is a powerful tool for nuclear biology to dissect the function of individual or gene pairs in synthetic, lethal situations.

An Erythrocyte Vesicle Protein Exported by the Malaria Parasite Promotes Tubovesicular Lipid Import from the Host Cell Surface

Plasmodium falciparum is the protozoan parasite that causes the most virulent of human malarias. The blood stage parasites export several hundred proteins into their host erythrocyte that underlie modifications linked to major pathologies of the disease and parasite survival in the blood. Unfortunately, most are ‘hypothetical’ proteins of unknown function, and those that are essential for parasitization of the erythrocyte cannot be ‘knocked out’. Here, we combined bioinformatics and genome-wide expression analyses with a new series of transgenic and cellular assays to show for the first time in malaria parasites that microarray read out from a chemical perturbation can have predictive value. We thereby identified and characterized an exported P. falciparum protein resident in a new vesicular compartment induced by the parasite in the erythrocyte. This protein, named Erythrocyte Vesicle Protein 1 (EVP1), shows novel dynamics of distribution in the parasite and intraerythrocytic membranes. Evidence is presented that its expression results in a change in TVN-mediated lipid import at the host membrane and that it is required for intracellular parasite growth, but not invasion. This exported protein appears to be needed for the maintenance of an essential tubovesicular nutrient import pathway induced by the pathogen in the host cell. Our approach may be generalized to the analysis of hundreds of ‘hypothetical’ P. falciparum proteins to understand their role in parasite entry and/or growth in erythrocytes as well as phenotypic contributions to either antigen export or tubovesicular import. By functionally validating these unknowns, one may identify new targets in host–microbial interactions for prophylaxis against this major human pathogen.

Towards an improved agricultural extension service as a key role player in the settlement of new farmers in South Africa

South Africa (SA) finds itself in a region where the rich countries perceive it as a sustainable and viable proposition for investment. The prospects for the future are bright but a warning light is burning: high food prices; shortage of essential food and the import of it; 50% of land reform projects have failed. Can the SA Agricultural Extension service play a role to address these issues? Research in SA clearly indicates a new concept of Extension and identifies 13 essential principles underlying any Extension approach, ranging from an educational and pro-active approach to an advisory and reactive approach. By means of consultation, discussion and dialogue extension practitioners and trainers developed an Agricultural Landscape for Extension, identifying specific concepts, study fields and essential knowledge/skills areas that form the “playing field” for the extension agent. An effective extension service is based on certain fixed principles namely: · The interrelationship between agricultural development and human development; · Development being needs based; · Participation being essential for all role players; and · Any intervention program being focused on behaviour change.The Landscape highlights the following concepts as being necessary to improve Agricultural Extension: - Technical competency (the extension agent must be an expert in at least one field of agriculture); - Communication skills (verbal; non verbal; written and mass communication); - Group facilitation skills (group dynamics and leadership); and - Extension management (program planning, management functions, ethics. monitoring and evaluation and leadership development). Land reform in SA is a priority program with efficient productivity as its ultimate outcome. The settlement of new farmers and specifically post settlement support become decisive. Guidelines to support the extension agent are: · The selection of new farmers by means of an effective screening instrument; · The farmer, or group of beneficiaries, need to work cohesively. The new farmer must have a clear understanding of the farm as a business; · Where possible the new farmer should be linked to a mentor; · The farm needs a viable and sustainable business plan; · The farm and farmer need financial support as prescribed in the business plan; and · The production of produce needs to be market driven. To-day's clients demand a professional service. The professional registration of extension agents should in own interest and in the interest of the industry be implemented as a matter of urgency. Extension in SA needs a vision that is focused on the future. Keywords : Extension landscape, principles, farmer settlement, technical competency South African Journal of Agricultural Extension Vol. 37 2008: pp. 58-84