A Plastid Protein That Evolved from Ubiquitin and Is Required for Apicoplast Protein Import in Toxoplasma gondii

Abstract

Apicomplexan parasites cause a variety of important infectious diseases, including malaria, toxoplasma encephalitis, and severe diarrhea due to Cryptosporidium. Most apicomplexans depend on an organelle called the apicoplast which is derived from a red algal endosymbiont. The apicoplast is essential for the parasite as the compartment of fatty acid, heme, and isoprenoid biosynthesis. The majority of the approximate 500 apicoplast proteins are nucleus encoded and have to be imported across the four membranes that surround the apicoplast. Import across the second outermost membrane of the apicoplast, the periplastid membrane, depends on an apicoplast-specific endoplasmic reticulum-associated protein degradation (ERAD) complex and on enzymes of the associated ubiquitination cascade. However, identification of an apicoplast ubiquitin associated with this machinery has long been elusive. Here we identify a plastid ubiquitin-like protein (PUBL), an apicoplast protein that is derived from a ubiquitin ancestor but that has significantly changed in its primary sequence. PUBL is distinct from known ubiquitin-like proteins, and phylogenomic analyses suggest a clade specific to apicomplexans. We demonstrate that PUBL and the AAA ATPase CDC48AP both act to translocate apicoplast proteins across the periplastid membrane during protein import. Conditional null mutants and genetic complementation show that both proteins are critical for this process and for parasite survival. PUBL residues homologous to those that are required for ubiquitin conjugation onto target proteins are essential for this function, while those required for polyubiquitination and preprotein processing are dispensable. Our experiments provide a mechanistic understanding of the molecular machinery that drives protein import across the membranes of the apicoplast.

The endoplasmic reticulum chaperone PfGRP170 is essential for asexual development and is linked to stress response in malaria parasites

“…To determine the role of PfGRP170 in trafficking proteins to the apicoplast, we removed TMP from PfGRP170-GFP-DDD parasites and examined the localisation of the apicoplast-localised cpn60 (Fellows, Cipriano, Agrawal, & Striepen, 2017;Florentin et al, 2017;Sheiner et al, 2011). No defects in apicoplast localisation of cpn60 were observed ( Figure 3c).…”

A mutagenesis screen for essential plastid biogenesis genes in human malaria parasites

“…ATG8's novel apicoplast function was discovered serendipitously by its unexpected localization on the apicoplast instead of autophagosomes (Kitamura et al, 2012). Though candidate approaches have yielded new molecular insight (Biddau et al, 2018;Fellows et al, 2017;Sheiner et al, 2015), in general they are indirect and may bias against novel pathways.…”

Evidence that disruption of apicoplast protein import in malaria parasites evades delayed-death growth inhibition

“…Over a dozen proteins have been implicated in import of nuclear-encoded apicoplast proteins, but of particular interest are proteins related to established drug targets in other systems. For example, the apicoplast-localized AAA ATPase CDC48 is related to mammalian p97 and is likely involved in translocation of apicoplast cargo across the periplastid membrane (12). Notably, mammalian p97, which plays an important role in ERAD and other cellular processes, is of interest as an anti-cancer drug target.…”

“…Most apicoplast proteins are 1) synthesized on cytosolic ribosomes, 2) trafficked to the apicoplast via the endoplasmic reticulum (ER), and 3) translocated across the 4 apicoplast membranes. This multistep import pathway involves more than a dozen proteins, including homologs of the translocation and ubiquitylation machinery typically involved in ER-associated degradation (ERAD) (8-12) as well as homologs of the TOC and TIC machinery found in plant plastids (9, 13-15).…”