Entry of newly synthesized GLUT4 into the insulin-responsive storage compartment is GGA dependent.
Following biosynthesis, both GLUT1 and VSV-G proteins appear rapidly (2-3 h) at the plasma membrane, whereas GLUT4 is retained in intracellular membrane compartments and does not display any significant insulin responsiveness until 6-9 h. Surprisingly, the acquisition of insulin responsiveness did not require plasma membrane endocytosis, as expression of a dominant-interfering dynamin mutant (Dyn/K44A) had no effect on the insulin-stimulated GLUT4 translocation. Furthermore, expression of endocytosis-defective GLUT4 mutants or continuous surface labeling with an exofacial specific antibody demonstrated that GLUT4 did not transit the cell surface prior to the acquisition of insulin responsiveness. The expression of a dominant-interfering GGA mutant (VHS-GAT) had no effect on the trafficking of newly synthesized GLUT1 or VSV-G protein to the plasma membrane, but completely blocked the insulin-stimulated translocation of newly synthesized GLUT4. Furthermore, in vitro budding of GLUT4 vesicles but not GLUT1 or the transferrin receptor was inhibited by VHS-GAT. Together, these data demonstrate that following biosynthesis, GLUT4 directly sorts and traffics to the insulin-responsive storage compartment through a specific GGA-sensitive process.
Watson
Robert T
RT
Department of Pharmacological Sciences, SUNY-Stony Brook, Stony Brook, NY 11794-8651, USA.
Khan
Ahmir H
AH
Furukawa
Megumi
M
Hou
June Chunqiu
JC
Li
Lin
L
Kanzaki
Makoto
M
Okada
Shuichi
S
Kandror
Konstantin V
KV
Pessin
Jeffrey E
JE
D041721:3T3-L1 Cells; D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism*; D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D034281:Dynamins / Q000235:genetics / Q000378:metabolism; D018274:Electroporation; D004705:Endocytosis / Q000502:physiology; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D007328:Insulin / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D022161:Transport Vesicles / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism
D033942:Adaptor Proteins, Vesicular Transport; C028391:G protein, vesicular stomatitis virus; C407306:GGA adaptor proteins; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D007328:Insulin; D008562:Membrane Glycoproteins; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D011993:Recombinant Fusion Proteins; C495678:Slc2a1 protein, mouse; C495693:Slc2a4 protein, mouse; D014759:Viral Envelope Proteins; D020727:ADP-Ribosylation Factors; D034281:Dynamins