| Acyl coenzyme A: cholesterol acyltransferase (ACAT) catalyzes the formation of intracellular cholesteryl esters in various tissues. Recently, human ACAT cDNA has been cloned and expressed in this laboratory (Chang, CC, et al, 1993, Cheng, D. 1995). We have also obtained rabbit polyclonal antibodies against the N-terminal portion of the ACAT protein (Chang, CC, et al, 1995). In this dissertation, I have studied the subcellular localization, turn-over and oligomerization of ACAT. Using indirect immunofluorescence, confocal microscopy, and subcellular fractionation, I showed that ACAT is mainly localized in the endoplasmic reticulum. I have successfully over-expressed human ACAT in wild-type Chinese hamster ovary (CHO) cells. While the ACAT protein is very stable, with a half life of more than 20 hours in human melanoma cells, the over-expression of ACAT in wild-type CHO cells leads to quick degradation of a portion of the ACAT protein (half life of less than one hour). This quick degradation in the CHO cells generates a 35 kDa intermediate fragment. We hypothesize that there may exist a rate-limiting intermediary step(s), possibly involving protein oligomerization in the ER, that leads to the normal processing of the mature ACAT protein.;We analyzed the hydrodynamic properties of ACAT. When solubilized with 1% deoxycholate, the sedimentation coefficient of ACAT protein-detergent complex is 9.6 S; its Stokes radius is 11.9 nm; its partial specific volume is 7.6 mg/cm;Cross-linking experiments with dithiobis-succinimidyl propionate (DSP) in vivo in melanoma cells shows that ACAT protein forms a complex of about 200 kDa in SDS-PAGE. Coimmunoprecipitation data in infected insect Sf9 cells show that ACAT can form a homo-oligomer suggesting that the ACAT complex may be a homo-tetramer. |
