The Journal of Cell Biology, Vol 108, 1257-1269, Copyright © 1989 by The Rockefeller University Press
Reconstitution of the Golgi apparatus after microinjection of rat liver Golgi fragments into Xenopus oocytes
J Paiement, M Jolicoeur, A Fazel and JJ Bergeron
Departement d'anatomie, Universite de Montreal, Quebec, Canada.
We have studied the reconstitution of the Golgi apparatus in vivo using an
heterologous membrane transplant system. Endogenous glycopeptides of rat
hepatic Golgi fragments were radiolabeled in vitro with [3H]sialic acid
using detergent-free conditions. The Golgi fragments consisting of
dispersed vesicles and tubules with intraluminal lipoprotein-like particles
were then microinjected into Xenopus oocytes and their fate studied by
light (LM) and electron microscope (EM) radioautography. 3 h after
microinjection, radiolabel was observed by LM radioautography over yolk
platelet-free cytoplasmic regions near the injection site. EM
radioautography revealed label over Golgi stacked saccules containing the
hepatic marker of intraluminal lipoprotein-like particles. At 14 h after
injection, LM radioautographs revealed label in the superficial cortex of
the oocytes between the yolk platelets and at the oocyte surface. EM
radioautography identified the labeled structures as the stacked saccules
of the Golgi apparatus, the oocyte cortical granules, and the plasmalemma,
indicating that a proportion of microinjected material was transferred to
the surface via the secretion pathway of the oocyte. The efficiency of
transport was low, however, as biochemical studies failed to show extensive
secretion of radiolabel into the extracellular medium by 14 h with
approximately half the microinjected radiolabeled constituents degraded.
Vinblastine (50 microM) administered to oocytes led to the formation of
tubulin paracrystals. Although microinjected Golgi fragments were able to
effect the formation of stacked saccules in vinblastine-treated oocytes,
negligible transfer of heterologous material to the oocyte surface could be
detected by radioautography. The data demonstrate that dispersed fragments
of the rat liver Golgi complex (i.e., unstacked vesicles and tubules)
reconstitute into stacked saccules when microinjected into Xenopus
cytoplasm. After the formation of stacked saccules, reconstituted Golgi
fragments transport constituents into a portion of the exocytic pathway of
the host cell by a microtubule- regulated process.
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