Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, C024

Oral Communications

Phosphorylation regulates LAT4 function in response to anticipated food intake and dietary protein content

L. Oparija1,2, A. Rajendran1,2, N. Poncet1,2, F. Verrey1,2

1. Institute of Physiology, University of Zurich, Zurich, Switzerland. 2. Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, Zurich, Switzerland.

System L amino acid transporter LAT4 (SLC43A2) is a sodium-independent uniporter that transports branched chain- and few other essential amino acids. Deletion of LAT4 leads to a slight intrauterine growth retardation, postnatal growth failure and early death in mice, possibly due to defective amino acid (re)absorption. Since it is suggested, that essential amino acid uniporters like LAT4 may limit amino acid flux through epithelia, we hypothesize that posttranslational modifications such as phosphorylation could regulate LAT4 function. We investigated the potential functional role of LAT4 phosphorylation sites, previously detected by others using phosphoproteomics, by expressing (non-)phosphorylation mutants in X. laevis oocytes. The dose-dependent uptake of radiolabeled phenylalanine (n = 30), kinetics assessment and immunofluorescence revealed that mimicking the non-phosphorylated state of S274 leads to an increase in LAT4 affinity (p<0.001, ANOVA), surface membrane localization and thus improves transport function; in contrast, the same mutation of S297 severely reduced the surface expression and transport rate of LAT4 (p<0.001, ANOVA). To investigate these sites in vivo, we used new phosphosite-specific antibodies to test the phosphorylation of S274 and S297 by Western blot and immunofluorescence. To determine whether LAT4 function, expression and intracellular localization are subjected to diurnal rhythm and whether they show an adaptive response to changes in dietary protein content, we entrained mice with time restricted feeding using normal (18%, n = 5) and low (8%, n =5) protein diets and sacrificed them by cervical dislocation either at the start of the active phase (ZT12), when food intake is expected or at the beginning of the passive phase, 4 hours after food removal (ZT0). We observed that the phosphorylation of intestinal LAT4, but not its expression nor its localization are submitted to diurnal rhythm. At the time of expected food intake, phosphorylation on S274 was low (p<0.001, ANOVA) and restricted to patches of cells along the villi, whilst phosphorylation on S297 was increased (p<0.05, ANOVA). On the other hand, mice maintained under 8% diet had a lower expression level of intestinal LAT4 (p<0.01, ANOVA) that displayed an increased intracellular localization, a higher S274 phosphorylation level (p<0.001, ANOVA) with more even distribution along the villi and a decreased S297 phosphorylation (p<0.01, ANOVA). These results suggest that under normal protein diet, the expectation of food intake enhances LAT4 function, leading to a higher amino acid absorption capacity. Under low protein diet, LAT4 response to food entrainment is limited and its transport activity appears to be set continuously on a lower level. In conclusion, we identified phosphorylation as a novel regulatory mechanism for a basolateral amino acid uniporter.

Where applicable, experiments conform with Society ethical requirements