The exocytotic trafficking of TC10 occurs through both classical and nonclassical secretory transport pathways in 3T3L1 adipocytes.
To examine the structural determinants necessary for TC10 trafficking, localization, and function in adipocytes, we generated a series of point mutations in the carboxyl-terminal targeting domain of TC10. Wild-type TC10 (TC10/WT) localized to secretory membrane compartments and caveolin-positive lipid raft microdomains at the plasma membrane. Expression of a TC10/C206S point mutant resulted in a trafficking and localization pattern that was indistinguishable from that of TC10/WT. In contrast, although TC10/C209S or the double TC10/C206,209S mutant was plasma membrane localized, it was excluded from both the secretory membrane system and the lipid raft compartments. Surprisingly, inhibition of Golgi membrane transport with brefeldin A did not prevent plasma membrane localization of TC10 or H-Ras. Moreover, inhibition of trans-Golgi network exit with a 19 degrees C temperature block did not prevent the trafficking of TC10 or H-Ras to the plasma membrane. These data demonstrate that TC10 and H-Ras can both traffic to the plasma membrane by at least two distinct transport mechanisms in adipocytes, one dependent upon intracellular membrane transport and another independent of the classical secretory membrane system. Moreover, the transport through the secretory pathway is necessary for the localization of TC10 to lipid raft microdomains at the plasma membrane.
Watson
Robert T
RT
Department of Physiology and Biophysics, The University of Iowa, Iowa City, Iowa 52242, USA.
Furukawa
Megumi
M
Chiang
Shian-Huey
SH
Boeglin
Diana
D
Kanzaki
Makoto
M
Saltiel
Alan R
AR
Pessin
Jeffrey E
JE
D016475:3T3 Cells; D017667:Adipocytes / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001693:Biological Transport, Active; D002451:Cell Compartmentation; D005089:Exocytosis; D007328:Insulin / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D020741:rho GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D007328:Insulin; D011994:Recombinant Proteins; C503260:Rhoq protein, mouse; D020741:rho GTP-Binding Proteins