Localization of TGN38 to the trans-Golgi network: involvement of a cytoplasmic tyrosine-containing sequence

To evaluate this phenomenon further, T-G-G was expressed by transient transfection in mouse MOP-8 cells. MOP-8 cells are transformed with an originless polyoma virus and thus replicate to high copy number the expression vector used in these studies, pCDM8, which has the polyoma virus origin of replication (Muller et al., 1984) . Consistent with the expected high levels of protein expression, immunofluorescent labeling of T-G-G in transfected MOP-8 cells showed surface staining in 63% of expressing cells (versus only '~10% in NRK cell transfections). Of MOP-8 cells expressing T-G-G at the plasma membrane, nearly all showed dimming or complete disappearance of endogenous TGN38 staining of the TGN as compared to untransfected cells in the same microscope field (Fig. 9, a and b) . Weak staining of TGN38 at the plasma membrane was observed in some of these cells (Fig. 9 b) . Disappearance of endogenous TGN38 staining was also observed in cells overexpressing other TGNlocalized chimeras, such as T-T-G and ID5 (data not shown). However, neither disappearance nor dimming of endogenous TGN38 staining was seen in MOP-8 cells overexpressing Tac (Fig. 9, c and d) , demonstrating the specificity of this phenomenon for overexpression of TGN-localized chimeras. Moreover, overexpression of T-G-G had no effect on the intensity and pattern of staining of a cis-medial Golgi marker, mannosidase II (Fig. 9 , e and f). One possible explanation for these observations is that TGN-localized chimeric proteins compete with endogenous TGN38 for interaction with a specific, saturable localization mechanism. However, alternative explanations, such as complete disruption of the TGN or masking of the TGN38 epitope by overexpression of the chimeric proteins, have not been ruled out at this time. More definitive evidence for saturability of a TGN localization mechanism will first require a demonstration of the structural integrity of the TGN in overexpressing cells and a quantitative demonstration that endogenous TGN38 is displaced from the TGN to the cell surface or other intracellular compartments. In any case, this phenomenon demonstrates an additional specificity of the cytoplasmic determinant, which not only can confer TGN localization but also can affect the distribution of an endogenous TGN resident protein.