Mutant/mutation
In the paper multiple mutants are described that express NUP138, C-terminally tagged with different biotin ligases (see below). For comparison of various biotin ligases, nucleoporin 138 (Nup138) was used as the bait protein. The mutant described here expresses NUP138, C-terminally tagged with TurboID. It is shown that TurboID is an efficient in vivo proximity labeling tool in P. berghei (see also below). 

Protein (function)
The nucleoporin Nup138 (the number defines the predicted molecular mass in kDa as in most Nups) was recently identified as an FG repeat nucleoporin; the protein is characterized by 14 dispersed FG repeats across its central region and is the smallest of the known FG-Nups conserved across the Plasmodium genus

The nuclear pore complex (NPC) is a large macromolecular assembly of around 30 different proteins, so-called nucleoporins (Nups). Nups are classified into two major groups: Firstly, the intrinsically disordered Nups with frequent FG (phenylalanine-glycine) repeats that line the central channel and interact with translocating cargo complexes. Secondly scaffold Nups that make up the cylindrical architecture grouped around the central channel. Transmembrane (TM) Nups – NDC1, GP210 and POM121 – anchor the NPC within the nuclear membrane.
In P. falciparum during the intraerythrocytic developmental cycle 3–7 nuclear pores are present per nucleus in early ring stage parasites. As the parasite grows the number increases transiently up to 60 in the trophozoite before they are being distributed among the 16–32 daughter progeny. 
In Plasmodium only two putative Nup homologs had been identified: PBANKA_1445400 (SEC13, previously characterized in P. falciparum) and PBANKA_ 0416300. Failing to identify further Nups by primary sequence homology to yeast or human Nups,  the P. berghei genome was scanned for proteins containing FG di-amino-acid repeats. This screen revealed PBANKA_0416300 (Nup100), PBANKA_0107600, PBANKA_0417900, PBANKA_1140100 and PBANKA_1310200 as potential FG-Nups
In this paper the following NUPs are identified:
NUP138, PBANKA_0417900
NUP205, PBANKA_1140100
NUP221, PBANKA_0416300
NUP313, PBANKA_1310200
NUP637; PBANKA_0107600
NUP176, PBANKA_1365100
NUP269, PBANKA_1454600
NUP335, PBANKA_0807900
NUP390, PBANKA_0309200
NUP434 PBANKA_0309400

Phenotype
For comparison of various biotin ligases, nucleoporin 138 (Nup138) was used as the bait protein. The mutant described here expresses NUP138, C-terminally tagged with TurboID. It is shown that TurboID is an efficient in vivo proximity labeling tool in P. berghei.

Following confirmation of genomic plasmid integration, we first carried out immunofluorescence assays probing for the 3X-HA or Myc tag to verify the stable expression and correct localization of all Nup138 fusion proteins. All anti-tag assays detected the various fusion proteins at the nuclear periphery except for the wild-type control and the turboid-ha(nup138.PP) mutant. The data provide evidence for the correct targeting of the tagged 138 nucleoporins.

Among all mutant lines, only the nup138::turboid parasite highlighted a distinct streptavidin signal close to the nuclear DNA consistent with earlier observations, when using green fluorescent protein (GFP), for Nup138::GFP. TurboID is thus the sole biotin ligase that labels the NPC at physiological biotin levels present in mouse serum, ranging between 6 to 15 ng/mL. Ninety four percent of examined nup138::turboid-ha parasites displayed perinuclear labeling indicating a high integration efficiency in this parasite population typical of genetically engineered P. berghei.

Additional information
Since the adaptation of BioID/BirA* to label and reveal protein-protein interactions, there have been continued efforts to improve this tool's efficiency. To reduce nonspecific interactions (for example, caused by erroneous targeting in the cell due to the large size of BioID) and improve the kinetics for biotinylation, a smaller second-generation system known as BioID2 was engineered from the bacterial Aquifex aeolicus biotin ligase. This protein is 27 kDa in size (as it lacks the DNA-binding domain), requires lower biotin concentrations, and performs well in mammalian cell culture and Plasmodium systems (33) at 37°C. Since the inherent labeling radius of BioID2 is only about 10 nm, the inclusion of a 25-nm linker consisting of 13 repeats of GGGGS (G glycine, S serine) between bait protein and biotin ligase allows for a greater radius of labeling; this facilitates the detection of factors that may interact transiently or are beyond the range of the original system. Further evolution in BioID methodologies is represented by TurboID, a 35 kDa protein with 15 mutations compared to wild-type BirA; the protein produces efficient labeling of proteins in 10 min in the presence of 50 μm of biotin in mammalian cell culture. A smaller, 28 kDa miniTurboID version was achieved by deleting the N-terminal DNA binding domain.

Other mutants