The novel roles of choline transporter-like 1 and 2 in ethanolamine transport

Abstract

We examined a novel function of mammalian Choline-Transporter-Like proteins CTL1/SLC44A1 and CTL2/SLC44A2 in ethanolamine transport. We established two distinct ethanolamine transport systems of a high affinity (K1 = 55.6 - 66.5 μM), mediated by CTL1, and of a low affinity (K2 = 275 - 299 μM), mediated by CTL2. Both types of transport are Na+-independent and mediated in a pH dependent manner, as expected for ethanolamine/H+ antiporters. Primary human fibroblasts with separate frameshift mutations (M1= SLC44A1ΔAsp517 and M2= SLC44A1ΔSer126) are devoid of CTL1 ethanolamine transport but maintain unaffected CTL2 transport. The lack of CTL1 or CTL2 reduced the ethanolamine transport, the flux by the CDP-ethanolamine Kennedy pathway and PE synthesis. Overexpression of CTL1 in SLC44A1ΔSer126 (M2) cells improved the ethanolamine transport and PE synthesis. The SLC44A1ΔSer126 cells are reliant on CTL2 function and CTL2 siRNA almost completely abolished ethanolamine transport in the whole cells and mitochondria. Overexpression of CTL1 and CTL2 cDNAs increased ethanolamine transport in control and SLC44A1ΔSer126 cells. CTL1 and CTL2 facilitated mitochondrial ethanolamine uptake, but the transport mediated by CTL1 is predominant in the whole cells and mitochondria. These data firmly established that CTL1 and CTL2 are the first identified ethanolamine transporters in the whole cells and mitochondria, with intrinsic roles in de novo PE synthesis by the CDP-Etn Kennedy pathway and compartmentation of intracellular ethanolamine.SignificanceThe lack of Choline Transporter Like 1 (SLC44A1/CTL1) is the primary cause of a new neurodegenerative disorder with elements of childhood-onset parkinsonism and mitochondrial dysfunction. SLC44A2/CTL2 encodes the human neutrophil antigen 3, causes autoimmune hearing loss and Meniere’s disease, and has been recently identified as the main risk factor for thrombosis-the major cause of death in Covid-19 patients. Our investigation provides insights into the novel functions of CTL1 and CTL2 as intrinsic ethanolamine transporters. CTL1 and CTL2 are high and low affinity transporters, with direct roles in the membrane phospholipid synthesis. The work contributes to new knowledge for CTL1 and CTL2 independent transport functions and the optimization of prevention and treatment strategies in those various diseases.

Organelle-selective click labeling coupled with flow cytometry allows high-throughput CRISPR screening of genes involved in phosphatidylcholine metabolism

“…To confirm this, we first quantified natural choline extracted from cytoplasm using a choline oxidase-mediated coupled enzyme assay, which revealed that the loss of FLVCR1 dramatically reduced the amount of endogenous choline inside cells (by nearly 80%) (Figure 4D). Note that these phenotypes of FLVCR-KO (decrease in PC synthesis and choline uptake) are the same phenotypes associated with major choline transporter inhibition (Taylor et al, 2021). Moreover, we found that the lower PC labeling intensity in FLVCR1-KO cells was partially restored by re-expression of FLVCR1 and completely rescued by overexpression of SLC5A7 (a high affinity choline transporter specifically expressed in cholinergic neurons) (Okuda et al, 2000); whereas overexpression of PCYT1A had little effect (Figure 4E).…”

Regulation of Membrane Phospholipid Homeostasis in Neurodegenerative Diseases

“…PC remained constant due to reduced PC degradation by PSS1 and increased formation by PE methylation [7]. Furthermore, we recently discovered that CTL1 also transport ethanolamine for PE synthesis, and that disruption in the PE homeostasis and consequently involving other PLs is indicative of an intricate internal system to maintain membrane PL balance [72].…”

“…Recently, we identified a new role for CTL1/SLC44A1 and CTL2/SLC44A2 proteins in transporting ethanolamine for PE synthesis. Therefore, since CTL1/2 transport plasma membrane choline and ethanolamine, they directly control PC and PE homeostasis and their membrane ratio [72]. Both CTL1 and CTL2 additionally localize and transport choline and ethanolamine in the mitochondria yet their role in the mitochondria is not completely clear [72].…”