Uptake by COPI-coated vesicles of both anterograde and retrograde cargo is inhibited by GTP gamma S in vitro

作者：Nickel W; Malsam J; Gorgas K; Ravazzola M; Jenne N; Helms JB; Wieland FT^*

来源：Journal of Cell Science, 1998, 111: 3081-3090.

• COP-coated vesicle
• vesicular transport
• protein sorting
• anterograde and retrograde cargo
• biosynthetic protein transport

On the basis of the cell surface protein CD8 we have constructed reporter molecules for both anterograde and retrograde transport from the Golgi complex. The cytoplasmic tail of CD8 was exchanged by a construct comprising a hemagglutinin (HA) epitope, the C-terminal sequence of the viral protein E19 (containing a KKXX retrieval signal) followed by a myc epitope (CDS-LT), Due to this masking of the KKXX retrieval signal 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 isolated Golgi membranes can be proteolytically converted into an unmasked reporter molecule for retrograde transport (CD8-ST) in vitro, A CHO cell line stably expressing CDS-LT was generated and used for the isolation 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 sorting into coat protein COPI-coated vesicles of these different kinds of cargo on a comparative basis. COPI-coated vesicles were generated in 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 CDS-ST were packaged into COPI-coated vesicles. However, COPI-coated vesicles generated in the presence of the slowly hydrolyzable analogue of GTP, GTP gamma S contained strikingly lower amounts of CD8-LT and CD8-ST, While COPI-coated vesicles accumulated about 12-fold in the presence of GTP gamma S these vesicles together contained only one fifth of cargo compared to the few vesicles generated in the absence of GTP gamma S, These data indicate that cargo packaging into COPI-coated vesicles requires hydrolysis of GTP.

• 出版日期1998-10