Distribution of ER and Golgi markers after
velocity gradient sedimentation. (A) Interphase (nonsynchronized) HeLa
cell postnuclear supernatants were fractionated on glycerol gradients
and immunoblotted with antibodies against the Golgi marker
protein GPP130 and the ER marker protein p63. Both Golgi and ER marker
proteins were recovered on a sucrose cushion at the bottom of the
gradient. (B) Mitotic HeLa cell postnuclear supernatants were
fractionated and immunoblotted with antibodies against
Golgi marker proteins (GPP130 and giantin) and ER marker proteins
(calnexin and p63). The cells were blocked at mitosis with 0.5 μg/ml
nocodazole and collected by shake-off. In contrast to interphase, a
major portion of the mitotic Golgi was recovered in a slowly
sedimenting position near the top of the gradient. These fractions
lacked detectable ER. (C) Fractions resulting from separation of
mitotic postnuclear supernatants were also assayed by enzymatic
activity for the Golgi marker galactosyltransferase and the ER marker
glucose-6-phophatase. By activity assay also, mitotic Golgi, but not
mitotic ER, was recovered in a slowly sedimenting position.
Centrifugation in these experiments was for 30 min at 150,000 ×
g; similar results were obtained under a variety of
centrifugation conditions.