The amino acids upstream of NH(2)-terminal dileucine motif play a role in regulating the intracellular sorting of the Class III transporters GLUT8 and GLUT12

Abstract

The transport of glucose across cell membranes is mediated by a family of facilitative glucose transporters (GLUTs). The class III glucose transporters GLUT8 and GLUT12 both contain a similar [DE]XXXL[LI] dileucine sorting signal in their amino terminus. This type of dileucine motif facilitates protein trafficking to various organelles or to the plasma membrane via interactions with adaptor protein (AP) complexes. The [DE]XXXL[LI] motif in GLUT8 is thought to direct it to late endosomal/lysosomal compartments via its interactions with AP1 and AP2. Unlike GLUT8, the [DE]XXXL[LI] motif does not direct GLUT12 to a lysosomal compartment. Rather, GLUT12 resides in the Golgi network and at the plasma membrane. In a previous study, we found that exchanging the XXX (TQP) residues in GLUT8 with the corresponding residues in GLUT12 (GPN) resulted in a dramatic missorting of GLUT8 to the cell surface. We postulated that the XXX amino acids upstream of the dileucine motif in GLUT8 influence the degree of interaction between the [DE]XXXL[LI] motif and adaptor proteins. To further explore its trafficking mechanisms, we created mutant constructs to identify the role that each of the individual XXX amino acids has for regulating the intracellular sorting of GLUT8. Here we find that the XXX amino acids, specifically the position of a proline -2 from the dileucine residues, influence the affinity of APs for GLUT8 and GLUT12.

Figure 1. NH₂-termini of GLUT constructs

A. The amino terminus of both wild type murine GLUT8 and wild type murine GLUT12 contains a [DE]XXXL[LI] motif. The motif differs between GLUT8 and GLUT12 by containing different amino acids in the positions -1, -2, and -3 to the dileucine motif. B. The [DE]XXXL[LI] motif in the amino terminus of GLUT8 was mutated to the constructs shown in order to explore the role that these amino acids play in the distinct sorting of GLUT8 and GLUT12.

Figure 2. The amino acids -1, -2, and -3 to the dileucine motif affect the cellular localization of GLUT8

A. The amino acids -1, -2, and -3 to the dileucine motif of GLUT8 were individually mutated to the corresponding amino acids from GLUT12. HEK293 cells were transiently transfected with each GLUT and were stained and examined by immunofluorescence microscopy. Like wild type GLUT8, GLUT8-GQP-HA and GLUT8-TQN-HA localize to an intracellular compartment. The GLUT8-GPN-HA construct, which contains the amino acids found in GLUT12, localizes to the plasma membrane. Similarly, the GLUT8-TPP-HA construct localizes to the cell surface. Images were obtained using a 60× oil objective lens. B. The cell surface expression of the mutant constructs GLUT8-TPP-HA and GLUT8-GPN-HA is significantly greater than that of wild type GLUT8-HA or the mutant constructs GLUT8-GQP-HA or GLUT8-TQN-HA (n=8).

Figure 3. The position of a proline residue -2 to the dileucine motif directs the localization of GLUT8 and GLUT12

A. To determine whether glutamine or proline affects the cellular localization of GLUT8, HEK293 cells were transfected and stained with an antibody directed against the HA-tag for GLUT8-HA, GLUT8-TAP-HA, GLUT8-APA-HA, and GLUT8-GPN-HA. Both GLUT8-HA and GLUT8-TAP-HA localized to an intracellular compartment, whereas GLUT8-APA-HA and GLUT8-GPN-HA trafficked to the cell surface. Images were obtained using a 60× oil objective lens. B. The amount of GLUT8-APA-HA and GLUT8-GPN-HA localized at the plasma membrane was significantly greater than that of GLUT8-HA or GLUT8-TAP-HA (n=8).

Figure 4. GLUT8 and the GLUT8TAP mutant localize to the late endosome/lysosome

HEK293 cells were transiently transfected with GLUTs and stained and examined by immunofluorescence microscopy. A. GLUT8 and GLUT8-TAP both colocalized with the late endosomal/lysosomal marker Syntaxin 8 (c, f). GLUT8-APA and GLUT8-GPN, which reside predominantly at the cell surface, did not colocalized with Syntaxin 8 (n=3) (i, l). Red channel: syntaxin 8 (a, d, g, j); Green channel: GLUT8 constructs (b,e,h,k); B. Both GLUT8 and GLUT8-TAP reside in the lysosomal membrane that surrounds the internalized FITC-dextran (n=3) (c, f); Red channel: FITC-dextran (a, d); Green channel: GLUT8 constructs (b, e). Images were obtained using a 60× oil objective lens.

Figure 5. GLUT8 and the GLUT8AAA mutant localize to the late endosome/lysosome

A. To determine whether threonine (-3) or proline (-1) affects the cellular localization of GLUT8, HEK293 cells were transfected and stained with an antibody directed against the HA-tag for GLUT8-HA, GLUT8-APA-HA, and GLUT8-AAA-HA. GLUT8-HA, GLUT8-TAP-HA, and GLUT8-AAA-HA localized to an intracellular compartment, whereas GLUT8-APA-HA trafficked to the cell surface. Images were obtained using a 60× oil objective lens. B. Both GLUT8-HA and GLUT8-AAA-HA (Green channel) colocalized with the late endosomal/lysosomal marker Syntaxin 8 (Red channel). (n=3).

Figure 6. Cell surface associated GLUT8 and GLUT8TAP are internalized and trafficked to an intracellular compartment

HEK293 cells were transiently transfected with GLUT8-HA, GLUT8-TAP-HA, GLUT8-APA-HA, or GLUT8-GPN-HA. At steady state, GLUT8-HA and GLUT8-TAP-HA predominantly reside in an intracellular compartment. GLUT8-APA-HA and GLUT8-GPN-HA reside in the plasma membrane. A monoclonal HA antibody was added to the cell culture media and incubated for one hour (1 hr), or washed out after one hour and cultured for an additional hour (2 hr). At both the one hour and two hour time points, GLUT8-HA and GLUT8-TAP-HA are internalized from the cell surface and redirected to an intracellular compartment. After one and two hours in culture, a majority of the labeled GLUT8-APA-HA and GLUT8-GPN-HA remains associated with the plasma membrane (n=3). Images were obtained using a 60× oil objective lens.

Figure 7. AP2 interacts with GLUT8 and the mutant GLUT8-TAP in HEK293 cells

A. The levels of RNA transcripts for the mu2 subunit of AP2 were knocked down by 91% in HEK293 cells by siRNA (n=3). B. RNAi treated HEK293 cells were transfected with GLUT8 or the GLUT8 mutants (n=3). The levels of GLUT8-HA and GLUT8-TAP-HA on the plasma membrane increased dramatically in cells transfected with RNAi targeting AP2. The cell surface levels of GLUT8-APA-HA and GLUT8-GPN-HA were not altered by the depletion of AP2. Images were obtained using a 60× oil objective lens.

Figure 8. AP1 interacts with GLUT8 and the mutant GLUT8-TAP in HEK293 cells

A. The transcript levels for the gamma1 subunit of AP-1 were depleted by 65% in HEK293 cells following treatment with siRNA (n=3). B. RNAi treated HEK293 cells were transiently transfected with GLUT8 or the GLUT8 mutants and stained without permeabilization (n=2). The cell surface associated GLUT8-HA and GLUT8-TAP-HA were greater in cells transfected with siRNA targeting AP1 than in cells transfected with negative control siRNA. The levels of GLUT8-APA-HA and GLUT8-GPN-HA at the plasma membrane were not altered by the depletion of AP1. Images were obtained using a 60× oil objective lens.