Yeast cells expressing DGAT1 proteins or vector control were collected by centrifugation and the pellet was washed once with ice-cold H2O containing 1 mM phenylmethylsulfonyl fluoride (PMSF). Microsome protein was prepared as described by Ro et al. (2002) (link). The microsome pellet was washed twice with ice-cold phosphate buffered saline (PBS) or ice-cold reaction buffer (RB, 20 mM Tris-HCl pH 7.6, 250 mM sucrose) and subjected for further protease protection assay or in vitro DGAT1 activity assay.
To enrich the reformed membrane sheets and provide access to luminal domains, the intact membrane compartments were agitated at 1 mg/ml in 200 mM Na2CO3, pH 11, with five passes through an insulin syringe as described by Wu et al. (2003) . The resuspended pellet was incubated on ice for 1 h. The unsealed membrane sample was collected, washed twice with ice-cold PBS and subjected for protease protection assay. Microsomal protein was quantified using Qubit® protein assay kit system (Life technologies, Q33211).
To enrich the reformed membrane sheets and provide access to luminal domains, the intact membrane compartments were agitated at 1 mg/ml in 200 mM Na2CO3, pH 11, with five passes through an insulin syringe as described by Wu et al. (2003) . The resuspended pellet was incubated on ice for 1 h. The unsealed membrane sample was collected, washed twice with ice-cold PBS and subjected for protease protection assay. Microsomal protein was quantified using Qubit® protein assay kit system (Life technologies, Q33211).