AK2

AK2 (PF3D7_0816900) is a monomeric 33 kDa adenylate kinase of Plasmodium falciparum that catalyzes the interconversion of ATP and AMP to ADP and is inhibited by P1,P5-(diadenosine-5')-pentaphosphate (PMID:18973776). Its N-terminus is N-myristoylated by P. falciparum N-myristoyltransferase (with which AK2 forms a heterodimer in vitro), a modification that stabilizes the enzyme (PMID:18973776), and together with a potential S-palmitoylation cysteine and a basic lysine cluster constitutes a targeting module: the N-terminal 37 residues alone are sufficient to direct AK2 to the parasite plasma membrane and drive its translocation across it into the parasitophorous vacuole (PMID:25909331). Downstream of the plasma membrane, AK2 is delivered to the parasitophorous vacuole membrane and tubovesicular network by an N-myristoylation-dependent, PEXEL-independent trafficking route: it is sorted at an ER subdomain by the PfArf1 GTPase toward the PV (PMID:33604307) and co-transported with PfRab5b to the tubovesicular network, with PfRab5b overexpression rerouting AK2 into punctate structures and blocking its arrival at the PVM (PMID:27316546, PMID:22819813). The physiological consequence of AK2 enzymatic activity at these membranes has not been characterized in the available corpus.

1. 2008 High

   Establishing that PF3D7_0816900 is a bona fide, monomeric adenylate kinase whose N-terminus is myristoylated by parasite NMT answered whether the protein is enzymatically active and post-translationally lipidated.

   Evidence Recombinant protein production, in vitro adenylate kinase assay with inhibitor characterization, co-expression with NMT in E. coli, mass spectrometry

   PMID:18973776

   Open questions at the time

   • Physiological substrate flux and role of AK2 activity in parasite energy metabolism not addressed
   • Functional significance of the AK2–NMT heterodimer in vivo unknown
2. 2012 Medium

   Localizing AK2 to the parasitophorous vacuole membrane and linking that localization to N-myristoylation answered where the enzyme resides and implicated its lipid modification in targeting.

   Evidence GFP-fusion protein expression and live cell fluorescence microscopy

   PMID:22819813

   Open questions at the time

   • Mechanism connecting myristoylation to PVM delivery not resolved at this stage
   • Single-lab GFP-fusion result without endogenous tag validation
3. 2015 Medium

   Dissecting the N-terminal targeting elements answered how AK2 is directed to and translocated across the plasma membrane, identifying a three-motif code.

   Evidence GFP-tagged variants and chimeric fusions, biochemical fractionation, fluorescence microscopy

   PMID:25909331

   Open questions at the time

   • Machinery that recognizes the palmitoylation and lysine motifs not identified
   • Mechanism of translocation across the plasma membrane not defined
4. 2016 Medium

   Demonstrating co-transport with PfRab5b from an ER-proximal compartment to the tubovesicular network answered which trafficking route carries AK2 beyond the plasma membrane.

   Evidence Co-expression with DD-tagged PfRab5b, selective membrane permeabilization, confocal microscopy, PfRab5b overexpression phenotype

   PMID:27316546

   Open questions at the time

   • Whether PfRab5b directly binds AK2 or acts indirectly not established
   • Cargo-recognition step at the ER not defined at this stage
5. 2021 Medium

   Identifying PfArf1 as the GTPase that sorts AK2 at the ER answered how AK2 enters the PV-directed, PEXEL-independent pathway and distinguished it from PfRab1b-dependent routes.

   Evidence Co-IP/MS of PfRab5b-associated proteins, constitutively active/inactive GTPase mutants, immunofluorescence and super-resolution microscopy

   PMID:33604307

   Open questions at the time

   • Direct physical interaction of PfArf1 with AK2 versus indirect effect not resolved
   • Molecular basis of AK2 recognition at the ER subdomain unknown

• The physiological purpose of positioning an adenylate kinase at the PVM/tubovesicular network and its requirement for parasite viability remain unresolved.
• No loss-of-function/essentiality data for AK2 in the corpus
• Local role of AK2 nucleotide interconversion at the PVM not tested
• No structural model of the targeting or catalytic domains