Redistribution of clathrin-coated vesicle adaptor complexes during adipocytic differentiation of 3T3-L1 cells.
Mechanisms for intracellular retention of proteins are induced during adipocytic differentiation of 3T3-L1 cells. To investigate the potential role of clathrin lattices in these retention processes, we performed a morphological and biochemical analysis of coated vesicle components in 3T3-L1 cells. Optical sectioning and image restoration revealed a marked increase in the staining of clathrin and beta adaptins in the perinuclear region of cells with differentiation. In addition, predominance of beta (subunit of the AP-2, plasma membrane adaptor) over beta' (subunit of the AP-1, Golgi adaptor) adaptin was observed in immunoblots of clathrin-coated vesicles purified from nondifferentiated fibroblasts, and this ratio was reversed in coated vesicles purified from differentiated adipocytes. These results indicate that the relative abundance of TGN-derived clathrin lattices increases markedly during adipocytic differentiation. Subcellular fractionation indicated that cytosolic AP-1 and AP-2 adaptors comprised approximately 70% of the total cellular adaptor pool. Interestingly, neither the concentration nor the relative ratio of cytosolic AP-1 to AP-2 adaptors increased significantly during differentiation. These data suggest that the increase in TGN-derived lattices results from differentiation-induced mechanisms for enhanced assembly or stabilization of adaptors on Golgi membranes. Interestingly, double-immunofluorescence microscopy also revealed that whereas extensive colocalization between clathrin and beta adaptins occurred both in fibroblasts and adipocytes, structures stained only with anti-adaptin antibody could be detected. Taken together these results suggest that membranes coated with adaptors, but not clathrin, can exist in these cells.
Chakrabarti
R
R
Program in Molecular Medicine, University of Massachusetts Medical School, Worcester 01655.
Joly
M
M
Corvera
S
S
D016475:3T3 Cells; D033965:Adaptor Protein Complex alpha Subunits; D033982:Adaptor Protein Complex gamma Subunits; D033942:Adaptor Proteins, Vesicular Transport; D017667:Adipocytes / Q000166:cytology / Q000378:metabolism*; D000818:Animals; D002451:Cell Compartmentation; D002454:Cell Differentiation; D002966:Clathrin / Q000302:isolation & purification; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism*; D051379:Mice; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D013347:Subcellular Fractions / Q000737:chemistry
D033965:Adaptor Protein Complex alpha Subunits; D033982:Adaptor Protein Complex gamma Subunits; D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; D011506:Proteins