Abstract
Golgi membranes and Golgi-derived vesicles are associated with multiple cytoskeletal proteins and motors, the diversity and distribution of which have not yet been defined. Carrier vesicles were separated from Golgi membranes, using an in vitro budding assay, and different populations of vesicles were separated using sucrose density gradients. Three main populations of vesicles labeled with beta-COP, gamma-adaptin, or p200/myosin II were separated and analyzed for the presence of actin/actin-binding proteins. beta-Actin was bound to Golgi cisternae and to all populations of newly budded vesicles. Centractin was selectively associated with vesicles co-distributing with beta-COP-vesicles, while p200/myosin II (non-muscle myosin IIA) and non-muscle myosin IIB were found on different vesicle populations. Isoforms of the Tm5 tropomyosins were found on selected Golgi-derived vesicles, while other Tm isoforms did not colocalize with Tm5 indicating the association of specialized actin filaments with Golgi-derived vesicles. Golgi-derived vesicles were shown to bind to F-actin polymerized from cytosol with Jasplakinolide. Thus, newly budded, coated vesicles derived from Golgi membranes can bind to actin and are customized for differential interactions with microfilaments by the presence of selective arrays of actin-binding proteins.
Highlights
Protein trafficking into and out of the Golgi complex requires multiple populations of membrane-bound vesicles or tubules
The budded vesicle pellet is enriched in p200/myosin II and -COP, compared with the original Golgi membranes, showing that coated vesicles have been generated during the assay and that these cytosolic proteins have bound to them (Fig. 1)
As an actin-based motor, p200/myosin II in the cytosol would be expected to bind to actin filaments; we found that 50% of the soluble p200/myosin II in the cytosol was pelleted along with the F-actin, under these conditions (Fig. 5)
Summary
Protein trafficking into and out of the Golgi complex requires multiple populations of membrane-bound vesicles or tubules. Using an in vitro assay to generate different populations of Golgiderived vesicles, we have analyzed the distinct arrays of actin, myosins II, tropomyosin isoforms, and other proteins associ- Vesicle budding is demonstrated by the de novo membrane binding of two cytosolic proteins, p200/myosin II and -COP (Fig. 1).
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