Abstract
The human L-type amino acid transporter 1 (LAT1), also known as SLC7A5, catalyzes the transport of large neutral amino acids across the plasma membrane. As the main transporter of several essential amino acids, notably leucine, LAT1 plays an important role in mTORC1 activation. Furthermore, it is overexpressed in various types of cancer cells, where it contributes importantly to sustained growth. Despite the importance of LAT1 in normal and tumor cells, little is known about the mechanisms that might control its activity, for example by promoting its downregulation via endocytosis. Here we report that in HeLa cells, activation of protein kinase C by phorbol 12-myristate 13-acetate (PMA) triggers efficient endocytosis and degradation of LAT1. Under these conditions we found LAT1 downregulation to correlate with increased LAT1 ubiquitylation. This modification was considerably reduced in cells depleted of the Nedd4-2 ubiquitin ligase. By systematically mutagenizing the residues of the LAT1 cytosolic tails, we identified a group of three close lysines (K19, K25, K30) in the N-terminal tail that are important for PMA-induced ubiquitylation and downregulation. Our study thus unravels a mechanism of induced endocytosis of LAT1 elicited by Nedd4-2-mediated ubiquitylation of the transporter’s N-terminal tail.
Highlights
The human L-type amino acid transporter 1 (LAT1), known as SLC7A5, catalyzes the transport of large neutral amino acids across the plasma membrane
We report that protein kinase C (PKC) activation by phorbol 12-myristate 13-acetate (PMA) induces rapid endocytosis and degradation of LAT1, that this downregulation coincides with increased ubiquitylation of LAT1, and that this modification involves the Nedd[] Ub ligase
We examined whether PMA-induced internalization of LAT1-GFP leads to its degradation
Summary
The human L-type amino acid transporter 1 (LAT1), known as SLC7A5, catalyzes the transport of large neutral amino acids across the plasma membrane. As the main transporter of several essential amino acids, notably leucine, LAT1 plays an important role in mTORC1 activation It is overexpressed in various types of cancer cells, where it contributes importantly to sustained growth. We report that in HeLa cells, activation of protein kinase C by phorbol 12-myristate 13-acetate (PMA) triggers efficient endocytosis and degradation of LAT1 Under these conditions we found LAT1 downregulation to correlate with increased LAT1 ubiquitylation. The transporters can potentially progress along the endocytic pathway and be delivered to the lysosome, where they are degraded This downregulation mechanism has been well studied in yeast, where ubiquitin (Ub) is the signal that generally triggers transporter endocytosis[1,2,3,4]. In agreement with prior predictions[38,39,40], the 12 transmembrane segments of LAT1 were found organized in a canonical LeuT fold[37]
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