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The notion of utilizing the binding affinity between UIM and UBL domains in the purification of 26S proteasomes have been approved by the purifications of mammalian 26S proteasomes from mouse muscle tissue and human embryonic kidney cells (Besche et al., 2009; Scanlon et al., 2009). Based on the affinity of PfUIM2 domain to the UBL domain of PfRad23, here we successfully adopted the affinity purification method to isolate the P. falciparum 26S proteasome complex from the parasite cell extracts. The use of PfUIM2 was due to the fact that it was shown to have the strongest affinity to both UBL domains and ubiquitin chains. To our knowledge, before this work there was no purification method available to purify the P. falciparum 26S proteasome. Li et al. reported that they have partially enriched the P. falciparum proteasome by using an anion exchange chromatography coupled to a size-exclusion chromatography (Li et al., 2012). Another pull down assay using an anti-GFP (anti-green fluorescent protein) antibody has immunoprecipitated nearly 30 proteasomal components associated with the GFP-tagged Rpn6 subunit from the P. falciparum whole extracts

(Muralidharan et al., 2011). However, such antibody-based purification methods require in vivo genetic modifications on a proteasomal subunit, which may have already perturbed the subunit associations (Wang et al., 2007). In great contrast, our affinity-based purification method offers a simple and robust method to isolate the intact P. falciparum 26S complex together with many proteasome-interacting proteins. Additionally, in vivo formaldehyde cross-linking was included to stabilize the assembly of the whole 26S complex as well as weak and transient interactions of some interactors with the plasmodial proteasome. Indeed, the use of a cross-linking strategy significantly increased the peptide recovery of the plasmodial 20S subunits and the number of co-purified proteins, compared to our preliminary tests without cross-linking.

The established purification method has allowed us to take the first insight into the integral composition of the P. falciparum 26S proteasome complex. The MS/MS analysis of the purified proteasome samples has revealed all putative subunits of the P. falciparum 26S proteasome. It is acknowledged that high throughput liquid chromatography-MS/MS data frequently suffer from a lack of reproducibility and identify very low abundant proteins. However, most of the P. falciparum proteasomal subunits were reproducibly and abundantly identified from four independent purifications, suggesting that they are indeed proteasomal components and were selectively co-purified within a complex. Interestingly, in the MS/MS analysis the number of observed peptides and sequence coverage for the plasmodial Rpn13 subunit appeared to be typically low, indicating a low abundance of Rpn13 among the co-purified proteasomal subunits. Consistently, similar results have been reported in several affinity purifications of 26S proteasomes from other organisms (Besche et al., 2009; Bousquet-Dubouch et al., 2009; Scanlon et al., 2009). These results are reminiscent of a recent finding that only one Rpn13 copy is present in a double-capped eukaryotic 26S proteasome, meaning a non-stoichiometric occupancy of Rpn13 in the majority of the 26S proteasome population (Berko et al., 2014). This could be a reasonable explanation for the low abundance of Rpn13 observed in the purified proteasome samples. Therefore, our data not only support this notion but also highly suggest a similar asymmetric integrity in the P. falciparum 26S proteasome. Besides, a plasmodial homolog of a proteasomal activator PA28 (in human PSMD10) was reproducibly identified, indicating its association with the plasmodial proteasome. In eukaryotes, three isoforms of PA28 subunits have been found. They form a heteroheptamer or hexamer complex that can attach to either one or both outer α-rings of the 20S CP, thus activating the peptidolytic activities of the 20S CP (Jung et al., 2013; Tomko et al., 2013). Besides, hybrid proteasomes with one 19S RP and one PA28 complex at opposite ends of the 20S CP (19S-20S-PA28) have also been identified (Hendil et al., 1998; Tanahashi et al., 2000). Accordingly, PA28 has been observed to be co-purified in other affinity-based purifications of eukaryotic proteasomes (Besche et al., 2009; Scanlon et al., 2009). Although the role of the PA28 complex has not been studied in P. falciparum, the identification of plasmodial PA28 subunits suggests that 19S-20S-PA28 proteasomes may exist in the parasites and that their role in the plasmodial 20S CP activation might be also conserved.

Three major peptidolytic activities of eukaryotic proteasomes have been detected in the purified proteasome samples, indicating that our purification method is able to isolate plasmodial proteasomes in an active form. When compared to the reported values of proteasomal activities, the activities of the purified plasmodial

proteasome determined in the eluted fractions was generally lower, although the measured chymotrypsin-like activity was comparable to the one of 26S proteasomes purified from human erythrocytes (a product of Enzo Life Science, PW8720). However, it should be mentioned that the reported values of eukaryotic proteasomal activities vary significantly depending on the purification method and the assay components. Due to the existence of different forms of proteasomes in eukaryotes (e.g. free 20S proteasomes, single or double-capped 26S proteasomes and hybrid proteasomes) and the inherently labile association between the 20S and the 19S subcomplexes (Tomko et al., 2013), different purification methods may result in a distinct composition of the isolated proteasomes. For example, conventional purifications of 20S proteasomes using multi-centrifugal procedures or affinity purifications via targeting the 20S subunits usually obtained a higher percentage of 20S proteasomes, which results in that the apparent proteasomal activities appear to be generally higher (Bousquet-Dubouch et al., 2009). In comparison, affinity purifications of 26S proteasomes via targeting to the 19S subunits may result in a certain extent of loss of the 20S due to the reasons that a certain amount of 20S may not associate with the 19S or they dissociate from the 19S during the purification procedure. Furthermore, it has been shown that affinity purifications of eukaryotic 26S proteasomes usually co-purify a number of additional proteins (Besche et al., 2009; Scanlon et al., 2009; Tai et al., 2010), which will be taken into account when calculating the proteasomal activities in the elution fractions. Besides the factor of purification method, different assay components appear to significantly influence proteasomal activities. For example, in some studies the proteasomal activities were measured within a buffer containing a proteasome-activating agent (SDS), which we did not use in our assays (Shibatani et al., 1995; Scanlon et al., 2009). Therefore, low proteasomal activities observed in the purified plasmodial proteasome samples may be due to the aforementioned factors, or even due to the origin itself.