An Interactome-Centered Protein Discovery Approach Reveals Novel Components Involved in Mitosome Function and Homeostasis in Giardia lamblia

Abstract

Protozoan parasites of the genus Giardia are highly prevalent globally, and infect a wide range of vertebrate hosts including humans, with proliferation and pathology restricted to the small intestine. This narrow ecological specialization entailed extensive structural and functional adaptations during host-parasite co-evolution. An example is the streamlined mitosomal proteome with iron-sulphur protein maturation as the only biochemical pathway clearly associated with this organelle. Here, we applied techniques in microscopy and protein biochemistry to investigate the mitosomal membrane proteome in association to mitosome homeostasis. Live cell imaging revealed a highly immobilized array of 30–40 physically distinct mitosome organelles in trophozoites. We provide direct evidence for the single giardial dynamin-related protein as a contributor to mitosomal morphogenesis and homeostasis. To overcome inherent limitations that have hitherto severely hampered the characterization of these unique organelles we applied a novel interaction-based proteome discovery strategy using forward and reverse protein co-immunoprecipitation. This allowed generation of organelle proteome data strictly in a protein-protein interaction context. We built an initial Tom40-centered outer membrane interactome by co-immunoprecipitation experiments, identifying small GTPases, factors with dual mitosome and endoplasmic reticulum (ER) distribution, as well as novel matrix proteins. Through iterative expansion of this protein-protein interaction network, we were able to i) significantly extend this interaction-based mitosomal proteome to include other membrane-associated proteins with possible roles in mitosome morphogenesis and connection to other subcellular compartments, and ii) identify novel matrix proteins which may shed light on mitosome-associated metabolic functions other than Fe-S cluster biogenesis. Functional analysis also revealed conceptual conservation of protein translocation despite the massive divergence and reduction of protein import machinery in Giardia mitosomes.

Giardia intestinalis mitosomes undergo synchronized fission but not fusion and are constitutively associated with the endoplasmic reticulum

“…This protein has been shown to function during the encystation process [55]. Indeed, we could show that its function is necessary for the completion of encystation, yet the presence of the dominant negative form of GlDRP did not affect the division of mitosomes, which is in contrast to the recent finding of Rout et al [70]. However, while our data suggest that dynamin-related proteins are not involved in mitosomal division, it is also possible that the level of the dominant negative form of dynamin capable of preventing encystation is not sufficient to interfere with mitosomal division.…”

Giardia lamblia: missing evidence for a canonical thioredoxin system

“…Since the search for a functional thioredoxin had remained unsuccessful, we performed a co-IP experiment for the isolation and identification of protein factors binding to TrxR following an established protocol (Rout et al 2016). We argued that a protein with thioredoxin function would likely be isolated together with TrxR.…”

“…Co-IP of proteins bound to TrxR was performed closely following an established protocol (Rout et al 2016) using immobilized anti-HA antibodies. However, cross-linking of proteins with Lamont's reagent was omitted.…”

“…Sample preparations for mass spectrometry, mass spectrometric identification of proteins and ensuing data analyses were performed as described recently (Rout et al 2016). …”

Phosphoinositide-binding proteins mark, shape and functionally modulate highly-diverged endocytic compartments in the parasitic protistGiardia lamblia

“…The Gl PXD4 interactome includes Gl CHC and Gl DRP and, uniquely for the Gl PXD protein family, a previously confirmed interaction with Gl PXD2 [19] albeit detected at lower stringencies (95_2_95, 2 hits) (Fig 4; Table S4). A putative SNARE protein GL50803_5785, previously identified in the Gl Tom40 interactome [42], was detected at lower stringencies (95_2_95, 2 hits). Similar to Gl PXD1, Gl PXD6 showed strong interaction with the β subunit of Gl AP2 and Gl CHC (Fig 4), although the reverse interaction was not detected in the previously-published clathrin-centered interactome [19].…”