UPTAKE BY COPI-COATED VESICLES OF BOTH ANTEROGRADE AND RETROGRADE CARGO IS INHIBITED BY GTP-GAMMA-S IN-VITRO

On the basis of the cell surface protein CD8 we have constructed repor ter molecules for both anterograde and retrograde transport from the G olgi complex. The cytoplasmic tail of CD8 was exchanged by a construct comprising a hemagglutinin (HA) epitope, the C-terminal sequence of t he viral protein E19 (containing a KKXX retrieval signal) followed by a myc epitope (CDS-LT), Due to this masking of the KKXX retrieval sign al CDS-LT is transported to the cell surface. Since the KKXX motif is joined to the myc epitope via a thrombin cleavage site, CD8-LT in isol ated Golgi membranes can be proteolytically converted into an unmasked reporter molecule for retrograde transport (CD8-ST) in vitro, A CHO c ell line stably expressing CDS-LT was generated and used for the isola tion of Golgi membranes. These membranes were shown to contain CDS-LT en route to the cell surface. By addition of thrombin, CDS-LT could be efficiently converted into CDS-ST, and this allows us to study the so rting into coat protein COPI-coated vesicles of these different kinds of cargo on a comparative basis. COPI-coated vesicles were generated i n vitro from Golgi membranes containing either CDS-LT or CDS-ST. When the incubation was performed in the presence of GTP, both CDS-LT and C DS-ST were packaged into COPI-coated vesicles. However, COPI-coated ve sicles generated in the presence of the slowly hydrolyzable analogue o f GTP, GTP gamma S contained strikingly lower amounts of CD8-LT and CD 8-ST, While COPI-coated vesicles accumulated about 12-fold in the pres ence of GTP gamma S these vesicles together contained only one fifth o f cargo compared to the few vesicles generated in the absence of GTP g amma S, These data indicate that cargo packaging into COPI-coated vesi cles requires hydrolysis of GTP.