Table 1.

Characterization of morphologically distinct GLUT4 compartments

Marker	Value	Compartment
		Vesicles	Tubules	Vacuoles
IRAP	% GLUT4 compartments positive for IRAP	87.5	86	97.5
	Ratio IRAP/GLUT4	0.84	0.53	0.79
EEA1	% GLUT4 compartments positive for EEA1	9.7	28.3	75.6
	Ratio EEA1/GLUT4	0.044	0.11	1.02
MPR46	% GLUT4 compartments positive for MPR46	56	75.9	95
	Ratio MPR46/GLUT4	0.41	0.5	0.56
VAMP-2	% GLUT4 compartments positive for VAMP-2	56.5	94.4	91.5
	Ratio VAMP-2/GLUT4	0.38	0.83	1.33

To analyze colocalization of GLUT4 with the indicated proteins, whole mount adipocyte rims were immuno-double-labeled, as described in Figure 4. Three different intracellular structures were defined by morphology: vesicles (between 60 and 100 nm in diameter), vacuoles (larger spherical structures), and tubules. For all randomly encountered structures gold particles corresponding to GLUT4 labeling and the indicated protein were counted. Only structures labeled for GLUT4 and counting at least two gold particles in total were considered. Compartments containing one or more gold particles for the indicated protein were considered positive for this marker (expressed as percentage of total GLUT4-positive compartments within this category). For the determination of the ratio, the sum of all gold particles counted for the indicated protein was divided by the sum of all gold particles for GLUT4 counted within the same category. For example, 9.7% of all GLUT4 positive vesicles also contained one or more gold particles for EEA1. The ratio for EEA1 to GLUT4 gold particles in these vesicles was as low as 0.044.