Oligomerization of a membrane protein correlates with its retention in the Golgi complex

Microsomes were prepared after metabolic labeling and the appropriate chase period from transfected HeLa cells plated in 6-cm dishes. Cells were rinsed in PBS and swelled in 0.8 ml swelling buffer (10 mM Tris, 15 mM NaCi, t mM MgCI2, pH 7.4) for 5 rain on ice. After scraping with a rubbet policeman, 0.25 ml swelling buffer containing 40% sucrose (wt/vol) was added, and the cells were homogenized in a I ml Dounce homogenizer with 60 strokes of the A pestle. Lysates were centrifuged briefly (90 s 5,000 rpm) to remove nuclei and large debris. 0.3 ml swelling buffer containing 10% sucrose (wt/vol) was added, and the lysates were centrifuged for 10 min at 100,000 rpm in a Beckman TL-100 ultracentrifuge (T100.2 rotor). Pellets were resuspended gently in PBS and divided into two aiiquots. TPCK-Trypsin (20/~g) was added to one aliquot, and both were incubated at 37"C for 30 min. Proteolytic digestion was stopped by adding 20 #g of soybean trypsin inhibitor to both tubes. Samples were solubilized (in detergent solution containing 0.4% SDS or in MNT as noted) and processed further as described in the text and figure legends.