GLUT8 Contains a [DE]XXXL[LI] Sorting Motif and Localizes to a Late Endosomal/Lysosomal Compartment

Abstract

Glucose transporter 8 (GLUT8) contains a cytoplasmic N-terminal dileucine motif and localizes to a thus far unidentified intracellular compartment. Translocation of GLUT8 to the plasma membrane (PM) was found in insulin-treated mouse blastocysts. Using overexpression of GLUT8 in adipocytes and neuronal cells however, insulin treatment or depolarization of the cells did not lead to GLUT8 PM translocation in other studies. In addition, other experiments showing dynamin-dependent endocytosis of GLUT8 suggested that GLUT8 recycles between an endosomal compartment and the PM. To reveal the functional/physiological role of GLUT8, we studied its subcellular localization in 3T3L1, HEK293 and CHO cells. We show that GLUT8 does not co-localize with GLUT4 and does not redistribute to the PM after treatment with insulin, ionophores or okadaic acid in these cell lines. Once endocytosed, GLUT8 does not recycle to the PM. GLUT8 localizes to late endosomes and lysosomes. An interspecies GLUT8 -sequence alignment revealed the presence of a highly conserved late endosomal/lysosomal-targeting motif ([DE]XXXL [LI]). Changing the glutamate to arginine as found in GLUT4 (RRXXXLL) alters GLUT8 endocytosis and retains the transporter at the PM. Furthermore, sorting GLUT8 to late endosomes/lysosomes does not require prior presence of GLUT8 at the PM followed by its endocytosis. In summary, GLUT8 does not reside in a recycling vesicle pool and is distinct from GLUT4. From our data, we postulate a role for GLUT8 in transport of hexoses across intracellular membranes, for example in specific compartments of GLUT8 expression such as the acrosome of mature spermatozoa or secretory granules in neurons. Furthermore, a role for GLUT8 in hexose transport across the lysosomal membrane, a transport mechanism that has long been suggested but unexplained, is discussed.

Identification and characterization of an alternatively spliced variant of the MHC class I-related porcine neonatal Fc receptor for IgG

“…Newlysynthesized proteins in the ER are transported to the trans-Golgi network (TGN) and then targeted either directly to endosomes or to the cell surface. The only pathways to the lysosome are characterized by way of early endosomes and then late endosomes usually following endocytosis and by way of late endosomes direct from the TGN [39,40]. It is well known that specific motifs, such as tyrosine-or dileucine-based motifs, in the cytoplasmic domain of proteins play a critical role in the intracellular trafficking of membrane proteins.…”

Structure of, and functional insight into the GLUT family of membrane transporters

“…175 Therefore, they hypothesized that GLUT8 transports glucose out of the rough endoplasmic reticulum into the cytosol to maintain cellular glucose homeostasis in neurons. 172,173 On the other hand, it has been observed that a highly conserved dileucine-containing motif (DEXXXLLI) was critical for GLUT8 sorting to the late endosomal/lysosomal compartments in mouse blastocytes.…”

“…172,173 In the case of MCF-7 cells, GLUT12 was localized perinuclearly when insulin was absent. 38 However, the acute change in subcellular distribution of GLUT12 was not detected after insulin treatments.…”

“…[170][171][172] Other targeting/trafficking studies showed that GLUT8 distributes more to the intracellular compartment in neurons and blastocysts than at the cell surface. [172][173][174] Piroli et al pointed out that, in rat hippocampal neurons, GLUT8 rapidly translocated to the rough endoplasmic reticulum following peripheral glucose administration. 175 Therefore, they hypothesized that GLUT8 transports glucose out of the rough endoplasmic reticulum into the cytosol to maintain cellular glucose homeostasis in neurons.…”

Functional Analysis of an Arabidopsis thaliana Abiotic Stress-inducible Facilitated Diffusion Transporter for Monosaccharides

“…2C, panel ii). GLUT8, which is a mammalian homologue of the ERD6-like family, is localized at the late endosome or the lysosome (35). GLUT8 contains an N-terminal acidic di-leucine motif EXXXL(L/I).…”