Plastid biogenesis in malaria parasites requires the interactions and catalytic activity of the Clp proteolytic system

Abstract

<jats:p>The human malaria parasite,<jats:italic>Plasmodium falciparum</jats:italic>, contains an essential plastid called the apicoplast. Most apicoplast proteins are encoded by the nuclear genome and it is unclear how the plastid proteome is regulated. Here, we study an apicoplast-localized caseinolytic-protease (Clp) system and how it regulates organelle proteostasis. Using null and conditional mutants, we demonstrate that the<jats:italic>P. falciparum</jats:italic>Clp protease (<jats:italic>Pf</jats:italic>ClpP) has robust enzymatic activity that is essential for apicoplast biogenesis. We developed a CRISPR/Cas9-based system to express catalytically dead<jats:italic>Pf</jats:italic>ClpP, which showed that<jats:italic>Pf</jats:italic>ClpP oligomerizes as a zymogen and is matured via transautocatalysis. The expression of both wild-type and mutant Clp chaperone (<jats:italic>Pf</jats:italic>ClpC) variants revealed a functional chaperone–protease interaction. Conditional mutants of the substrate-adaptor (<jats:italic>Pf</jats:italic>ClpS) demonstrated its essential function in plastid biogenesis. A combination of multiple affinity purification screens identified the Clp complex composition as well as putative Clp substrates. This comprehensive study reveals the molecular composition and interactions influencing the proteolytic function of the apicoplast Clp system and demonstrates its central role in the biogenesis of the plastid in malaria parasites.</jats:p>

A redox-active crosslinker reveals an essential and inhibitable oxidative folding network in the endoplasmic reticulum of malaria parasites

“…Control parental parasites (lacking HA-tagged Pf J2) were also used for immunoprecipitation and analyzed in the same manner. Each co-IP experiment was performed in triplicate, and the abundance of each identified protein was calculated by summing the total MS1 intensities of all matched peptides for each selected protein, and normalizing by the total summed intensity of all matched peptides in the sample ( Fig 3A ) [ 20 , 21 ]. Because Pf J2 is an ER-localized protein, we further filtered our list of interacting partners to those containing a signal peptide and/or at least one transmembrane domain (i.e.…”

“…The filter ensures that our list is comprised of proteins that potentially localize or traverse via the cellular compartment where Pf J2 is located. We identified a stringent list of biologically relevant interacting partners as those proteins which were present in all three Pf J2 apt coIP experiments, and were at least 5-fold more abundant compared to the controls, as previously described [ 21 ] ( Fig 3B and 3C ). A complete list of all identified proteins is provided in S1 Table .…”

Structure, function, and substrates of Clp AAA+ protease systems in cyanobacteria, plastids, and apicoplasts: A comparative analysis

“…This approach provides an alternative to chemical complementation with IPP to probe the essential apicoplast function. ClpP, ClpR, ClpC, and surprisingly also ClpS (ClpS is not essential for viability in plants, algae, cyanobacteria, and nonphotosynthetic bacteria) are each essential for apicoplast viability because they are required for apicoplast biogenesis, and the absence of Clp components led to growth arrest and apicoplast loss ( 53 , 55 ) ( Fig. 3 ).…”

“…3 ). Loss-of-function mutants for ClpP, ClpC, and ClpS could be obtained when grown in presence of IPP, allowing the introduction of modified ClpP and ClpC transgenes to probe Clp processing, assembly, protein–protein interactions, and trap substrates ( 53 , 55 ). ClpP and ClpR form each homoheptameric rings, but they do not appear to form a stable tetradecameric complex together ( 56 , 57 ).…”

Redesigned TetR-Aptamer System To Control Gene Expression in Plasmodium falciparum

“…Such tandem repeats are inherently unstable and tend to be deleted by recombination. We and others have observed truncations in the aptamer array with a consequent loss of control over gene expression, thereby limiting the utility of this knockdown strategy ( 30 , 31 , 36 , 37 , 43 ). To rectify this, we modified the array to contain unique spacers while retaining the secondary structure of the aptamers.…”