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Vol. 9, Issue 3, 623-635, March 1998
Department of Biological Sciences, Carnegie Mellon University,
Pittsburgh, Pennsylvania 15213
Partitioning of the mammalian Golgi apparatus during cell division
involves disassembly at M-phase. Despite the importance of the
disassembly/reassembly pathway in Golgi biogenesis, it remains unclear
whether mitotic Golgi breakdown in vivo proceeds by direct vesiculation
or involves fusion with the endoplasmic reticulum (ER). To test whether
mitotic Golgi is fused with the ER, we compared the distribution of ER
and Golgi proteins in interphase and mitotic HeLa cells by
immunofluorescence microscopy, velocity gradient fractionation, and
density gradient fractionation. While mitotic ER appeared to be a fine
reticulum excluded from the region containing the spindle-pole body,
mitotic Golgi appeared to be dispersed small vesicles that penetrated
the area containing spindle microtubules. After cell disruption,
M-phase Golgi was recovered in two size classes. The major breakdown
product, accounting for at least 75% of the Golgi, was a population of
60-nm vesicles that were completely separated from the ER using
velocity gradient separation. The minor breakdown product was a larger,
more heterogenously sized, membrane population. Double-label
fluorescence analysis of these membranes indicated that this portion of
mitotic Golgi also lacked detectable ER marker proteins. Therefore we
conclude that the ER and Golgi remain distinct at M-phase in HeLa
cells. To test whether the 60-nm vesicles might form from the ER at
M-phase as the result of a two-step vesiculation pathway involving
ER-Golgi fusion followed by Golgi vesicle budding, mitotic cells were
generated with fused ER and Golgi by brefeldin A treatment. Upon
brefeldin A removal, Golgi vesicles did not emerge from the ER. In
contrast, the Golgi readily reformed from similarly treated interphase
cells. We conclude that Golgi-derived vesicles remain distinct from the ER in mitotic HeLa cells, and that mitotic cells lack the capacity of
interphase cells for Golgi reemergence from the ER. These experiments suggest that mitotic Golgi breakdown proceeds by direct vesiculation independent of the ER.
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