Similar [DE]XXXL[LI] motifs differentially target GLUT8 and GLUT12 in Chinese hamster ovary cells

Abstract

The transport of glucose across cell membranes is mediated by facilitative glucose transporters (GLUTs). The recently identified class III GLUT12 is predominantly expressed in insulin-sensitive tissues such as heart, fat and skeletal muscle. We examined the subcellular localization of GLUT12 in Chinese hamster ovary and human embryonic kidney 293 cells stably expressing murine GLUT12. We have previously shown that another class III GLUT8 contains a [DE]XXXL[LI] motif that directs it to late endosomal/lysosomal compartments. Despite also having this highly conserved motif in its amino terminus, GLUT12 does not colocalize with GLUT8. Rather, GLUT12 resides in the Golgi network and at the plasma membrane (PM). Furthermore, GLUT8 and GLUT12 exhibit dramatic differences in trafficking from the PM. Whereas GLUT8 is internalized following its expression at the cell surface, GLUT12 remains largely associated with the PM. To further explore the trafficking mechanisms, we created mutant constructs to explore the potential role of GLUT12's NH(2)-terminal dileucine motif in regulating its intracellular sorting. We show that both the GPN and the LL residues within the [DE]XXXL[LI] motif influence the cell surface expression of GLUT12 and conclude that the mechanisms governing the intracellular sorting of GLUT12 are distinct from those regulating the sorting of GLUT8.

Figure 1. Western blot analysis of GLUT12

Whole cell lysates were prepared from HEK293 cells transiently transfected with GLUT12FLAG (lane 1 and 2), GLUT12 (lane 3), or empty vector (lane 4). In lane 1, GLUT12FLAG containing cell lysates were subjected to immunoprecipitation with anti-FLAG antibody. Lysates prepared from cells expressing GLUT12 were treated with (lane 6) or without PNGaseF (lane 5) to identify glycosylated and nonglycosylated band size. Proteins were separated by SDS-PAGE, transferred onto nitrocellulose, and probed with the anti-GLUT12 antibody at a dilution of 1:2000.

Figure 2. GLUT12 and GLUT8 reside in different compartments

CHO cells stably expressing GLUT8-GFP were transiently transfected with GLUT12. GLUT12 localizes both to a perinuclear compartment (A) and to the plasma membrane (B), but fails to colocalize with GLUT8. In endometrial stromal cells(C), both GLUT12 and GLUT8 are endogenously expressed. Subcellular fractionation confirms that GLUT12 is predominantly in the plasma membrane (PM) fraction whereas GLUT8 is distributed in the high and low density membrane fractions. Images were taken at 60× magnification.

Figure 3. GLUT12 localizes to the Golgi network

GLUT12 is detected in the Golgi network (A -GM130) and colocalizes with the post-Golgi membrane trafficking Rab11-YFP (B) in stably GLUT12-expressing HEK293 cells. GLUT12 does not colocalized with Syntaxin 8 or Rab7-YFP (C and D). Images were taken at 60× magnification.

Figure 4. The NH₂-terminal region of GLUT12 contains a highly conserved [DE]XXXL[LI] motif

(A) GLUT12 sequence comparison was performed using Clustal W to align the NH₂-terminus of GLUT12 from several species. Highly conserved residues in the motif are highlighted with red letters. (B) Similar [DE]XXXL[LI] motifs are also found in the lysosomal protein LIMPII and in the NH₂-terminal region of the Class III glucose transporter GLUT8. The alignment of EXXXLL with the rat dileucine motif is also shown. (C) In this study, the GLUT12 NH₂-terminal dileucine motif has been mutated to dialanine as shown in blue letters. In addition, the three amino acids between the acidic residue and the dileucine in the EXXXLL motif in GLUT12 were switched with the equivalent residues from the GLUT8 sequence. The reciprocal mutation in GLUT8 was also generated.

Figure 5. Mutating the EGPNLL motif of mouse GLUT12 to EGPNAA or to ETQPLL decreases the cell surface expression of GLUT12

(A). HEK293 cells were transiently transfected with GLUT12-HA, GLUT12-AA-HA, or GLUT12-TQP-HA. Cells were stained with or without prior permeabilization using an HA-antibody. Decreased levels of plasma membrane staining were observed in GLUT12-AA or GLUT12-TQP-expressing cells compared to the cell surface expression levels of wild-type GLUT12. (B) Relative amounts of cell surface-associated GLUT12 were quantified using flow cytometry with HEK293 cells transiently expressing GLUT12-HA, GLUT12-AA-HA, or GLUT12-TQP-HA. In agreement with the immunofluorescence results, GLUT12-AA-HA and GLUT12-TQP-HA exhibit less cell surface expression than wild-type GLUT12. (C) GLUT12-HA and GLUT12-TQP-HA localized to similar compartments in endometrial stromal cells which endogenously express GLUT12. Images were taken at 60× magnification.

Figure 6. Mutating the ETQPLL motif of mouse GLUT8 to EGPNLL retains the transporter at the plasma membrane

(A) HEK293 cells were transiently transfected with GLUT8-HA or with GLUT8-GPN-HA. Cells were stained with HA-antibody with or without permeabilization. In contrast to wild-type GLUT8, GLUT8-GPN primarily localizes to the plasma membrane. (B) The relative amounts of cell surface-associated GLUT8 were quantified using flow cytometry in HEK293 cells that were transiently transfected with GLUT8-HA or GLUT8-GPN-HA. The GLUT8-GPN-HA mutation increases the amount of protein at the cell surface, suggesting that the mutation may hinder GLUT8 endocytosis. (C) GLUT8-HA and GLUT8-GPN-HA localize to similar compartments in endometrial stromal cells which endogenously express GLUT8. Images were taken at 60× magnification.

Figure 7. Internalization of GLUT12 and retention of GLUT8 occur when substitutions are made to the XXXL[LI] residues

CHO cells were transiently transfected with GLUT12-HA, GLUT12-TQP-HA, GLUT8-HA, or GLUT8-GPN-HA. The steady state localization (0 hr) of the transfected GLUTs in CHO cells was determined by staining permeabilized (P) and nonpermeabilized (NP) cells with an HA antibody. Cells were cultured for one hour with the HA antibody (1 hr) or after rinsing out the antibody, cultured for an additional hour (2 hr). Cells with or without prior permeabilization were then incubated with Alexa-488 to label intracellular or plasma membrane HA antibody. (A) At steady state levels (0 hr), most of GLUT12 localizes to the plasma membrane, whereas a majority of GLUT12-TQP localizes to an intracellular compartment. Following one and two hours of cell surface exposure to an HA-antibody, the majority of GLUT12 remained associated with the cell surface. In contrast to GLUT12, much of the GLUT12-TQP was internalized within the two hours. (B) At steady state levels, the majority of GLUT8 localizes to an intracellular compartment. The GLUT8 that is associated with the cell surface becomes internalized after one and two hour time periods. GLUT8-GPN, on the other hand, resides primarily at the cell surface at its steady state distribution. Similar to GLUT12, most GLUT8-GPN remains associated with the cell surface one and two hours after exposure to an HA-antibody. Images were taken at 60× magnification.