To establish recycling routes in the early secretory pathway we have studied the recycling of the ER-Golgi intermediate compartment (ERGIC) marker ERGIC-53 in HepG2 cells. Immunofluorescence microscopy showed progressive concentration of ERGIC-53 in the Golgi area at 15 degreesC. Upon rewarming to 37 degreesC ERGIC-53 redistributed into the cell periphery often via tubular processes that largely excluded anterograde transported albumin. Immunogold labeling of cells cultured at 37 degreesC revealed ERGIC-53 predominantly in characteristic beta-COP-positive tubulo-vesicular clusters both near the Golgi apparatus and in the cell periphery. Concentration of ERGIC-53 at 15 degreesC resulted from both accumulation of ERGIC-53 in the ERGIC and movement of ERGIC membranes closer to the Golgi apparatus. Upon rewarming to 37 degreesC the labeling of ERGIC-53 in the ERGIC rapidly returned to normal levels whereas ERGIC-53's labeling in the cis-Golgi was unchanged. Temperature manipulations had no effect on the average number of ERGIC-53 clusters. Density gradient centrifugation indicated that the surplus ERGIC-53 accumulating in the ERGIC at 15 degreesC was rapidly transported to the ER upon rewarming. These results suggest that the ERGIC is a dynamic membrane system composed of a constant average number of clusters and that the major recycling pathway of ERGIC-53 bypasses the Golgi apparatus.

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