Volume 6 issue 12 December 2005
GLUT8 contains a [DE]XXXL[LI] sorting motif and localizes to a late endosomal/lysosomal compartment.
Robert Augustin, Joan Riley, Kelle H. Moley.

SUPLEMENTARY MATERIAL:

Supplemental Figure 1: Endocytosed GLUT4 continuously recycles between its intracellular location and the plasma membrane.
Transiently GLUT4-myc transfected CHO-cells were stained for intracellular or surface expression of GLUT4 using the myc-antibody with (a) or without prior permeabelization (d), respectively. GLUT4-myc transfected cells were cultured for one hour with the HA-antibody (b, e) or, after washing away the antibody, kept in culture for another hour (c, f). Cells with (b, c) or without prior (e, f) permeabilization were then incubated with an anti mouse Alexa 488 antibody to label the internalized HA-antibody. Surface expression of GLUT4 remains the same among cells that are: 1) stained for plasma membrane GLUT4 without prior permeabilization (d) 2) cultured for one hour with the myc-antibody (e) and 3) cultured for 1 hour with the myc antibody and then incubated for an additional hour after washing out the antibody (f).

Supplementary figure 1 (.tif)

Supplemental Figure 2: GLUT8-RR remains at the plasma membrane in stably transfected cell but also shows an intracellular localization that overlaps with LAMP1.
Two stably GLUT8-HA expressing CHO-cell clones were stained for cell surface GLUT8 using the HA-antibody recognizing exclusively cell surface GLUT8 in non-permeabilized cells. Staining of permeabilized cells for GLUT8 (red) and LAMP1 (green) shows colocalization of the two proteins (Bar indicates 10µm) .

Supplementary figure 2 (.tif)

Supplemental Figure 3: Mutation of the amino acids QE or E in the EXXXLL motif of GLUT8 to alanine results in increased PM expression and altered intracellular distribution of the mutated proteins compared to wildtype GLUT8.
CHO-cells were transiently transfected with either GLUT8-HA, GLUT8-A-HA or GLUT8-AA-HA and the PM expression as well as intracellular expression was examined by confocal microscopy in non-permeabilized (a, c, e) and permeabelized cells (b, d, f), respectively. (Bar indicates 10µm).

Supplementary figure 2 (.tif)