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Genetic evidence that an endosymbiont-derived endoplasmic reticulum-associated protein degradation (ERAD) system functions in import of apicoplast proteins - PubMed

  • ️Thu Jan 01 2009

Genetic evidence that an endosymbiont-derived endoplasmic reticulum-associated protein degradation (ERAD) system functions in import of apicoplast proteins

Swati Agrawal et al. J Biol Chem. 2009.

Abstract

Most apicomplexan parasites harbor a relict chloroplast, the apicoplast, that is critical for their survival. Whereas the apicoplast maintains a small genome, the bulk of its proteins are nuclear encoded and imported into the organelle. Several models have been proposed to explain how proteins might cross the four membranes that surround the apicoplast; however, experimental data discriminating these models are largely missing. Here we present genetic evidence that apicoplast protein import depends on elements derived from the ER-associated protein degradation (ERAD) system of the endosymbiont. We identified two sets of ERAD components in Toxoplasma gondii, one associated with the ER and cytoplasm and one localized to the membranes of the apicoplast. We engineered a conditional null mutant in apicoplast Der1, the putative pore of the apicoplast ERAD complex, and found that loss of Der1(Ap) results in loss of apicoplast protein import and subsequent death of the parasite.

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Figures

FIGURE 1.
FIGURE 1.

T. gondii ERAD components are associated with the ER/cytoplasm and the apicoplast. a–g, immunofluorescence analysis of parasites transfected with the coding sequences of seven putative T. gondii ERAD proteins carrying a C-terminal epitope tag (g, the Ufd1Ap epitope tag was constructed by inserting the tag directly into the genomic locus, see

supplemental methods

). We also expressed Cdc48Ap as a recombinant protein and raised a specific antiserum. Localization of the native protein detected with this antibody is indistinguishable from the protein derived from the tagged transgene (Fig. 2 and

supplemental Fig. S1

). Insets in the merge in panels e–g show a 2-fold higher magnification. h–j, Western blot analysis of apicoplast ERAD components. As for most apicoplast proteins, two bands are apparent (p, precursor; m, mature protein (9, 36)). ACP, luminal apicoplast marker, HDEL, T. gondii ER marker P30-GFP-HDEL (35).

FIGURE 2.
FIGURE 2.

The ERAD components Der1Ap and Cdc48Ap localize to the membrane compartment surrounding the apicoplast. Host cells infected with a transgenic T. gondii line expressing an HA-tagged version of Der1Ap were fixed, frozen, and sectioned with a cryo-ultramicrotome. Sections were incubated with a rat antibody to HA (a, b, d, black arrowheads), a rabbit serum to ACP (9) (a and b, white arrowhead), and a newly developed rabbit antibody against recombinant Cdc48Ap (c and d, white arrowhead with black outline, see also

supplemental Fig. S1

) followed by secondary antibody conjugated to 12 or 18 nm colloidal gold, stained with uranyl acetate/methylcellulose, and analyzed by transmission electron microscopy. Panel b shows a higher magnification of the cell shown in panel a, the four membranes of the apicoplast are indicated by black arrowheads outlined in white. Ap, apicoplast; Go, golgi; Nu, nucleus.

FIGURE 3.
FIGURE 3.

Disruption of the native Der1Ap locus. a, Der1Ap locus was disrupted by homologous recombination using a targeting construct carrying a CAT marker flanked by 2 kb of gene-specific upstream and downstream sequences. Recombinants were isolated using chloramphenicol to select for CAT expression, and cell sorting to select against YFP expression. Wild type (RH-WT), parental (carrying the native (Der1Ap) as well as an inducible minigene version (iDer1Ap), and ΔDer1Ap (carrying only the inducible version) were tested for the presence of the native locus by b, PCR (position of primers is indicated in a), or c, Southern blot using the radiolabeled coding sequence of Der1Ap as probe and Nde1 restriction to distinguish the native and inducible locus.

FIGURE 4.
FIGURE 4.

Der1Ap is essential for parasite growth. a–c, fluorescence growth assays for the ΔDer1Ap mutant carrying the inducible copy only (a), the parental strain carrying both inducible and native Der1Ap (b), and a ΔDer1Ap clone complemented with the Der1Ap gene driven by a constitutive promoter (c, all strains were engineered to express RFP). Assays were performed in triplicate (error bars reflect S.D., note that the error bar is only shown if larger than the symbol (>3%)) in the absence (circles) and presence (squares) of 0.5 μ

m

ATc or after 3 days of ATc preincuabtion (triangles). d–f, plaque assays measuring impact of loss of Der1AP. Confluent HFF cultures were infected with 400 parasites of the ΔDer1Ap mutant (d), parental (e), or complemented (f) strain, respectively, and cultured for 9 days in the absence (−ATc) or presence (+ATc) of anhydrous tetracycline. Cultures were fixed and stained as described under “Materials and Methods.” Note loss of growth in the mutant under ATc that is restored by gene complementation.

FIGURE 5.
FIGURE 5.

Genetic ablation of Der1Ap results in loss of apicoplast protein import and organellar defects. a, Western blot analysis of Der1Ap-HA levels in ΔDer1Ap in response to ATc treatment (the dense granule protein GRA8 serves as loading control). b, pulse-chase (P/C) analysis of post-translational modification of apicoplast (FNR-RFP, Cpn60, PDH-E2), mitochondrial (mito(E2)), and secretory (MIC5) proteins. The data shown are representative of three independent experiments. Bands labeled with an asterisk likely represent RFP expression from an internal translation start site, mitochondrial Cpn60, and human PDH-E2 (12), respectively. Note that ATc treatment in parental parasites has no effect on apicoplast protein import (see

supplemental Fig. S2

and Ref. 12) c, phosphorimager quantification of mature apicoplast-targeted protein bands shown in b. d, number of “four parasite” vacuoles in which each parasite has a clearly discernable apicoplast (n = 100 for each data point, see

supplemental Fig. S3

for additional detail).

FIGURE 6.
FIGURE 6.

Divergent origins of T. gondii Cdc48 proteins. RAxML maximum likelihood tree derived from an alignment of Cdc48 proteins from 30 taxa (900 unambiguously aligned amino acid characters were used after manual inspection, GenBank™ accession numbers are provided in the

supplemental methods

). Bootstrap analyses were conducted using 100 replicates. Nm, nucleomorph (remnant endosymbiont nucleus), Pl, plastid, Ap, apicoplast, Cy, cytoplasm.

FIGURE 7.
FIGURE 7.

Multiple distinct translocons act in apicoplast protein import. a, Der1, Cdc48, and Ufd1 are believed to be core components of the translocon of the ERAD pathway. b, duplication of the Der1 gene in the algal endosymbiont and relocation of its protein product to its plasma membrane enables c, protein import from the host endomembrane system. d, schematic representation of trafficking of nuclear-encoded proteins to the lumen of the apicoplast comparing a model employing subsequent Der1, Toc, and Tic translocons with a model in which the Toc translocon has been subsequently replaced by a second Der1 translocon. Cargo proteins are shown in yellow. The transit peptide (purple) is cleaved upon arrival. Note that trafficking of certain apicoplast membrane proteins appears not to require a bipartite signal sequence (26) and might occur through a different mechanism.

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