Co-operative function and mutual stabilization of the half ATP-binding cassette transporters HAF-4 and HAF-9 in Caenorhabditis elegans

Abstract

Caenorhabditis elegans HAF-4 and HAF-9 are half ABC (ATP-binding-cassette) transporters that are highly homologous to the human lysosomal peptide transporter TAPL [TAP (transporter associated with antigen processing)-like; ABCB9]. We reported previously that both HAF-4 and HAF-9 localize to the membrane of a subset of intestinal organelles, and are required for the formation of these organelles and other physiological aspects. In the present paper, we report the genetic and physical interactions between HAF-4 and HAF-9. Overexpression of HAF-4 and HAF-9 did not rescue the intestinal organelle defect of the haf-9 and haf-4 deletion mutants respectively, indicating that they cannot substitute for each other. Double haf-4 and haf-9 mutants do not exhibit more severe phenotypes than the single mutants, suggesting their co-operative function. Immunoprecipitation experiments demonstrated their physical interaction. The results of the present study suggest that HAF-4 and HAF-9 form a heterodimer. Furthermore, Western blot analysis of the deletion mutants and RNAi (RNA interference) knockdown experiments in GFP (green fluorescent protein)-tagged HAF-4 or HAF-9 transgenic worms suggest that HAF-4-HAF-9 heterodimer formation is required for their stabilization. The findings provide a clue as to how ABC transporters adopt a stable functional form.

Compatibility between mitochondrial and nuclear genomes correlates with the quantitative trait of lifespan in Caenorhabditis elegans

“…The number of worms was counted (about 300 adults), and the worms were then washed 3 times with M9 buffer (22 mM KH2PO4, 42 mM Na2HPO4, 85 mM NaCl, 1 mM MgSO4) containing 0.1% Tween-20 to remove E. coli OP50 (all but 500 μL of M9 buffer was removed each time from the worm pellet). Worms were then lysed as described previously 49 . ATP was measured using a luciferase based assay (Sigma, product number FL-AA), using the manufacturer’s protocol.…”

ABC-B transporter genes in Dirofilaria immitis

“…Cel-HAF-4 and Cel-HAF-9 were localized on the membranes of intestinal granular organelles ( Kawai et al., 2009 ). Cel-HAF-4 and Cel-HAF-9 were reported to form a heterodimer that was required for their own stabilization ( Tanji et al., 2013 ). They both were reported to be necessary for a normal defecation cycle, for proper growth and for normal brood size ( Kawai et al., 2009 ).…”

Characterization of HAF-4- and HAF-9-localizing organelles as distinct organelles in Caenorhabditis elegans intestinal cells

“…Bristol strain N2 was used as the standard wild-type strain, and the following mutant and transgenic strains were obtained from the Caenorhabditis Genetics Center (University of Minnesota): haf-4(ok1042) , haf-4(gk240) , haf-9(gk23) , lmp-1(nr2045) , glo-1(zu391) , rab-7(ok511) , hjIs67[atgl-1::GFP + mec-7::mRFP] , bIs1[vit-2::GFP + rol-6(su1006)] , hjIs9[ges-1p::glo-1::GFP + unc-119(+)] , hjIs37[vha-6p::mRFP-PTS1 + Cbr-unc-119(+)] , hjIs8[ges-1p::GFP-PTS1] , and zcIs17[ges-1::GFP(mit)] . The transgenic strains Is[haf-4::GFP + rol-6(su1006)] , Is[haf-9::GFP + rol-6(su1006)] , Is[haf-9::mCherry + rol-6(su1006)] , and Is[ges-1p::lmp-1::mRFP + rol-6(su1006)] were established as described by Kawai et al [ 17 ] and Tanji et al [ 18 ]. Is[dhs-3::GFP] was provided by Dr. Liu (Institute of Biophysics, Chinese Academy of Sciences, Beijing, China).…”

“…Previously, we reported that HAF-4 and HAF-9, ATP-binding cassette (ABC) transporter proteins that are homologous to the mammalian lysosomal peptide transporter TAPL (transporter associated with antigen processing like, ABCB9), localize to the membrane of a subset of intestinal granular organelles approximately 2 μm in diameter from the late larval to adult stages [ 17 ]. HAF-4 and HAF-9 are involved in maintaining the normal formation of their localizing organelles, probably by forming a heterodimer as a functional unit of a transporter [ 17 , 18 ]. The colocalization of HAF-4, HAF-9, and LMP-1, a homolog of mammalian lysosome-associated membrane proteins (LAMPs), suggests their relevance to lysosomes.…”