Role of the N-terminal transmembrane domain in the endo-lysosomal targeting and function of the human ABCB6 protein.

ABCB6 is a homodimeric ATP-binding cassette (ABC) transporter present in the plasma membrane and in intracellular organelles. The intracellular localization of ABCB6 has been a matter of debate, as it has been suggested to reside in the mitochondria and the endo-lysosomal system. Using a variety of imaging modalities including confocal and electron microscopy we confirm the endo-lysosomal localization of ABCB6 and show that the protein is internalized from the plasma membrane through endocytosis, to be distributed to multivesicular bodies and lysosomes. In addition to the canonical nucleotide binding (NBD) and transmembrane domains (TMD), ABCB6 contains a unique N-terminal transmembrane domain (TMD0), which does not show sequence homology to known proteins. We investigated the functional role of these domains through the molecular dissection of ABCB6. We find that the folding, dimerization, membrane insertion and ATP binding/hydrolysis of the core ABCB6 complex devoid of TMD0 is preserved. However, in contrast to the full-length transporter, the core ABCB6 construct is retained at the plasma membrane, and does not appear in Rab5-positive endosomes. TMD0 is directly targeted to the lysosomes, without a passage to the plasma membrane. Collectively, our results reveal that TMD0 represents an independently folding unit, which is dispensable for catalysis, but has a crucial role in the lysosomal targeting of ABCB6

Shifting the Paradigm: The Putative Mitochondrial Protein ABCB6 Resides in the Lysosomes of Cells and in the Plasma Membrane of Erythrocytes

ABCB6, a member of the adenosine triphosphate–binding cassette (ABC) transporter family, has been proposed to be responsible for the mitochondrial uptake of porphyrins. Here we show that ABCB6 is a glycoprotein present in the membrane of mature erythrocytes and in exosomes released from reticulocytes during the final steps of erythroid maturation. Consistent with its presence in exosomes, endogenous ABCB6 is localized to the endo/lysosomal compartment, and is absent from the mitochondria of cells. Knock-down studies demonstrate that ABCB6 function is not required for de novo heme biosynthesis in differentiating K562 cells, excluding this ABC transporter as a key regulator of porphyrin synthesis. We confirm the mitochondrial localization of ABCB7, ABCB8 and ABCB10, suggesting that only three ABC transporters should be classified as mitochondrial proteins. Taken together, our results challenge the current paradigm linking the expression and function of ABCB6 to mitochondria

ABCB6 is not present in the purified mitochondrial fraction of K562 cells.

<p>Cellular membranes were separated by differential centrifugation (A); mitochondria were isolated using anti-TOM22 magnetic beads (B) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037378#s4" target="_blank">Materials and Methods</a>. Following separation by SDS-PAGE, organelle membranes were transferred to a PVDF membrane, which was incubated in antibodies recognizing specific organelle markers and ABCB6. <b>A</b>. Cellular fractions corresponding to total cell lysate (lane 1); nuclei and intact cells (lane 2); mitochondrial pellet of the 8000×g centrifugation (lane 3); pellet of 12.000×g centrifugation (lane 4); pellet of 20.000×g centrifugation (lane 5). <b>B</b>. Isolation of mitochondria using TOM22 immunobeads. Total cell lysate (lane 1); fraction bound to the beads after repeated washing steps (lane 2); pellet of the flow through (lane 3).</p

Human reticulocytes release ABCB6 in association with exosomes. A

<p>. 250 µL packed cells volume (PCV) of human RBCs from reticulocyte-enriched (4.5%) blood was cultured for 48 h and exosomes were collected from the medium as previously described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037378#pone.0037378-Barres1" target="_blank">[42]</a>. 0.5 µL PCV of RBCs before (t0) or after (48 h) maturation, as well as the completeness of exosomes were loaded on 10% SDS-PAGE for immunoblot analysis of the indicated proteins. The molecular mass (kDa) standards are indicated on the left. Note that the TfR is not detected in RBCs due to the low reticulocyte count but revealed in exosomes due to its concentration in the vesicles. <b>B</b>. Human RBCs from reticulocyte-enriched (10%) blood were adsorbed on cover slips pretreated with poly-L-lysine, fixed 20 min by 1% PFA and immunostained for ABCB6 (antibody Ab 7474 at; dilution 1/:50/Alexa Fluor 594; A21207) or and TfR (MoAb H68.4 at; dilution 1/:200/Alexa Fluor 488; A11029) after permeabilization, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037378#s4" target="_blank">Materials and Methods</a>. Coverslips were observed using a Leica confocal SPE and a Leica 63× ACS APO 1.33 objective. Note that none of the cells shown are positive to DAPI although contained in the mounting reagent. Arrows point to possible colocalization of ABCB6 and TfR in MVE.</p

Interventions to improve system-level coproduction in the Cystic Fibrosis Learning Network

Background Coproduction is defined as patients and clinicians collaborating equally and reciprocally in healthcare and is a crucial concept for quality improvement (QI) of health services. Learning Health Networks (LHNs) provide insights to integrate coproduction with QI efforts from programmes from various health systems.Objective We describe interventions to develop and maintain patient and family partner (PFP) coproduction, measured by PFP-reported and programme-reported scales. We aim to increase percentage of programmes with PFPs reporting active QI work within their programme, while maintaining satisfaction in PFP-clinician relationships.Methods Conducted in the Cystic Fibrosis Learning Network (CFLN), an LHN comprising over 30 cystic fibrosis (CF) programmes, people with CF, caregivers and clinicians cocreated interventions in readiness awareness, inclusive PFP recruitment, onboarding process, partnership development and leadership opportunities. Interventions were adapted by CFLN programmes and summarised in a change package for existing programmes and the orientation of new ones. We collected monthly assessments for PFP and programme perceptions of coproduction and PFP self-rated competency of QI skills and satisfaction with programme QI efforts. We used control charts to analyse coproduction scales and run charts for PFP self-ratings.Results Between 2018 and 2022, the CFLN expanded to 34 programmes with 52% having ≥1 PFP reporting active QI participation. Clinicians from 76% of programmes reported PFPs were actively participating or leading QI efforts. PFPs reported increased QI skills competency (17%–32%) and consistently high satisfaction and feeling valued in their work.Conclusions Implementing system-level programmatic strategies to engage and sustain partnerships between clinicians and patients and families with CF improved perceptions of coproduction to conduct QI work. Key adaptable strategies for programmes included onboarding and QI training, supporting multiple PFPs simultaneously and developing financial recognition processes. Interventions may be applicable in other health conditions beyond CF seeking to foster the practice of coproduction