PKC-dependent endocytosis of the GLT1 glutamate transporter depends on ubiquitylation of lysines located in a C-terminal cluster.
The activity of the main glutamate transporter in the CNS, GLT1, can be regulated by protein kinase C (PKC). It is known that activation of PKC by phorbol esters promotes the clathrin-dependent internalization of the transporter, followed by its lysosomal degradation. However, the molecular mechanisms that link PKC activation and the internalization of GLT1 are not fully understood. In this article, we show that this internalization process is dependent on the ubiquitylation of lysine residues located in the C-terminal tail of GLT1. Exposure to PMA increases the ubiquitylation of GLT1 in transfected cells and in the rat brain, and this ubiquitylated GLT1 accumulates in the intracellular compartment. However, internalization of ubiquitylated GLT1 was blocked with a dominant negative dynamin 2 mutant, indicating that the addition of ubiquitin moieties to the transporter in the membrane precedes its endocytosis. The elimination of lysines from the C-terminus of the transporter (lysines 497, 517, 526, 550, 558, 570, and 573) blocked GLT1 ubiquitylation and endocytosis. However, reintroduction of lysine 517 alone into this mutant was sufficient to restore PMA dependent ubiquitylation and internalization of GLT1. Similarly, reintroduction of lysine 526 restored the endocytosis, while this was only partially recovered after the individual reintroduction of lysines 550 or 570. These data suggest that the activation of PKC induces the ubiquitylation of these C-terminal lysine residues in GLT1 and that this modification mediates the interaction of the transporter with the endocytic machinery.
González-González
Inmaculada M
IM
Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Consejo Superior de Investigaciones Científicas and CIBERER, Madrid, Spain.
García-Tardón
Noemí
N
Giménez
Cecilio
C
Zafra
Francisco
F
D020816:Amino Acid Motifs / Q000235:genetics / Q000502:physiology; D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004285:Dogs; D004705:Endocytosis / Q000235:genetics / Q000502:physiology*; D027342:Excitatory Amino Acid Transporter 2 / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D011493:Protein Kinase C / Q000235:genetics / Q000502:physiology*; D051381:Rats; D054875:Ubiquitination / Q000235:genetics / Q000502:physiology*
D027342:Excitatory Amino Acid Transporter 2; D010446:Peptide Fragments; D011493:Protein Kinase C; D008239:Lysine