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

Oligomerization of the Gml protein was analyzed by velocity gradient centrifugetion in sucrose performed essentially as described (Doms et al., 1987) . Continuous 5-20% (wt/wt) sucrose gradients were poured over a 60% (wt/wt) sucrose cushion (0.4 nil) in SW50.1 tubes. All solutions were in MNT (100 mM NaCI, 20 mM Tris, 30 mM MES, pH 5.8) containing 0.1% Triton X-100. HeLa cells expressing either VSV G or Gml were metabolically labeled for 5 min and harvested (in MNT containing 1% Triton X-100) either immediately or after 60 min of chase. Lysates were loaded on top of the gradients and spun at 44-45,000 rpm for 15-16 h. Fractions (0.35 ml) were collected from the top using a Buchler Auto Densi-Flow IIC, immunoprecipitated with anti-VSV antibody, and electrophoresed to determine the location of the proteins in the gradient. For estimating the size of the oligomer, gradients (5-20% sucrose [wt/wt] in 100 mM NaC1, 50 mM Tris, pH 5.8, 0.1% Triton X-100) were spun at 47000 rpm for 4 h. The markers used were thyroglobulin (19.3 $20,,0) and eatalase (11.3 $20,,0).