FIGURE 8.

Formation of Sec22-Sec22-HA(L213C) homodimers in vesicle fusion reactions demonstrates luminal compartment mixing. Two-stage transport reactions in which COPII vesicles containing [³⁵S]gpαf were synthesized from microsomes expressing Sec22(L213C) in CBY773 and mixed with Golgi acceptor membranes containing Sec22HA(L213C) expressed in CBY773. Reactions were incubated at the indicated temperatures for 60 min in the presence of an ATP regeneration system and (+) or (−) the LMA1 and Uso1 fusion factors (Recon) to reconstitute transport. EGTA (3 mm) was added to reactions as indicated. The bar graph plots the amount of Golgi-modified [³⁵S]gpαf to reflect vesicle fusion. Immunoblots of parallel reactions were probed with antibodies against Sec22 and Sly1 (loading control) to monitor the level of Sec22-Sec22HA mixed dimer formation.