Our current understanding of mitochondrial functioning is largely restricted to traditional model organisms, which only represent a fraction of eukaryotic diversity. The unusual mitochondrion of malaria parasites is a validated drug target but remains poorly understood. Here, we apply complexome profiling to map the inventory of protein complexes across the pathogenic asexual blood stages and the transmissible gametocyte stages of Plasmodium falciparum. We identify remarkably divergent composition and clade-specific additions of all respiratory chain complexes. Furthermore, we show that respiratory chain complex components and linked metabolic pathways are up to 40-fold more prevalent in gametocytes, while glycolytic enzymes are substantially reduced. Underlining this functional switch, we find that cristae are exclusively present in gametocytes. Leveraging these divergent properties and stage dynamics for drug development presents an attractive opportunity to discover novel classes of antimalarials and increase our repertoire of gametocytocidal drugs.

No abstract

Our current understanding of mitochondrial functioning is largely restricted to traditional model organisms, which only represent a fraction of eukaryotic diversity. The unusual mitochondrion of malaria parasites is a validated drug target but remains poorly understood. Here, we apply complexome profiling to map the inventory of protein complexes across the pathogenic asexual blood stages and the transmissible gametocyte stages of Plasmodium falciparum. We identify remarkably divergent composition and clade-specific additions of all respiratory chain complexes. Furthermore, we show that respiratory chain complex components and linked metabolic pathways are up to 40-fold more prevalent in gametocytes, while glycolytic enzymes are substantially reduced. Underlining this functional switch, we find that cristae are exclusively present in gametocytes. Leveraging these divergent properties and stage dynamics for drug development presents an attractive opportunity to discover novel classes of antimalarials and increase our repertoire of gametocytocidal drugs.

No abstract

Complexome profiling (CP) is a state-of-the-art approach that combines separation of native proteins by electrophoresis, size exclusion chromatography or density gradient centrifugation with tandem mass spectrometry identification and quantification. Resulting data are computationally clustered to visualize the inventory, abundance and arrangement of multiprotein complexes in a biological sample. Since its formal introduction a decade ago, this method has been mostly applied to explore not only the composition and abundance of mitochondrial oxidative phosphorylation (OXPHOS) complexes in several species but also to identify novel protein interactors involved in their assembly, maintenance and functions. Besides, complexome profiling has been utilized to study the dynamics of OXPHOS complexes, as well as the impact of an increasing number of mutations leading to mitochondrial disorders or rearrangements of the whole mitochondrial complexome. Here, we summarize the major findings obtained by this approach; emphasize its advantages and current limitations; discuss multiple examples on how this tool could be applied to further investigate pathophysiological mechanisms and comment on the latest advances and opportunity areas to keep developing this methodology.

Despite the enormous significance of malaria parasites for global health, some basic features of their ultrastructure remain obscure. In this study, we apply high-resolution volumetric electron microscopy to examine and compare the ultrastructure of Plasmodium falciparum gametocytes of both genders and in different stages of development as well as the more intensively studied asexual blood stages revisiting previously described phenomena in 3D. In doing so, we challenge the widely accepted notion of a single mitochondrion by demonstrating the presence of multiple mitochondria in gametocytes. We also provide evidence for a gametocyte-specific cytostome variant. Furthermore, we generate, among other organelles, the first 3D reconstructions of endoplasmic reticulum (ER), Golgi apparatus, and exported structures in gametocytes. Assessing interconnectivity between organelles, we find frequent structural appositions between the nucleus, mitochondria, and apicoplast. We provide evidence that the ER is a promiscuous interactor with numerous organelles and the trilaminar membrane of the gametocyte. Public availability of these volumetric electron microscopy resources of wild-type asexual and sexual blood-stage malaria parasites will facilitate reinterrogation of this global dataset with different research questions and expertise. Taken together, we reconstruct the 3D ultrastructure of P. falciparum gametocytes in high detail and shed light on the unique organellar biology of these deadly parasites.

Complexome profiling is an emerging ‘omics approach that systematically interrogates the composition of protein complexes (the complexome) of a sample, by combining biochemical separation of native protein complexes with mass-spectrometry based quantitation proteomics. The resulting fractionation profiles hold comprehensive information on the abundance and composition of the complexome, and have a high potential for reuse by experimental and computational researchers. However, the lack of a central resource that provides access to these data, reported with adequate descriptions and an analysis tool, has limited their reuse. Therefore, we established the ComplexomE profiling DAta Resource (CEDAR, www3.cmbi.umcn.nl/cedar/), an openly accessible database for depositing and exploring mass spectrometry data from complexome profiling studies. Compatibility and reusability of the data is ensured by a standardized data and reporting format containing the “minimum information required for a complexome profiling experiment” (MIACE). The data can be accessed through a user-friendly web interface, as well as programmatically using the REST API portal. Additionally, all complexome profiles available on CEDAR can be inspected directly on the website with the profile viewer tool that allows the detection of correlated profiles and inference of potential complexes. In conclusion, CEDAR is a unique, growing and invaluable resource for the study of protein complex composition and dynamics across biological systems.

Complexome profiling allows large-scale, untargeted, and comprehensive characterization of protein complexes in a biological sample using a combined approach of separating intact protein complexes e.g., by native gel electrophoresis, followed by mass spectrometric analysis of the proteins in the resulting fractions. Over the last decade, its application has resulted in a large collection of complexome profiling datasets. While computational methods have been developed for the analysis of individual datasets, methods for large-scale comparative analysis of complexomes from multiple species are lacking. Here, we present Comparative Clustering (CompaCt), that performs fully automated integrative analysis of complexome profiling data from multiple species, enabling systematic characterization and comparison of complexomes. CompaCt implements a novel method for leveraging orthology in comparative analysis to allow systematic identification of conserved as well as taxon-specific elements of the analyzed complexomes. We applied this method to a collection of 53 complexome profiles spanning the major branches of the eukaryotes. We demonstrate the ability of CompaCt to robustly identify the composition of protein complexes, and show that integrated analysis of multiple datasets improves characterization of complexes from specific complexome profiles when compared to separate analysis. We identified novel candidate interactors and complexes in a number of species from previously analyzed datasets, like the emp24, the V-ATPase and mitochondrial ATP synthase complexes. Lastly, we demonstrate the utility of CompaCt for the automated large-scale characterization of the complexome of the mosquito Anopheles stephensi shedding light on the evolution of metazoan protein complexes. CompaCt is available from https://github.com/cmbi/compact-bio.