Effect of microtubule assembly status on the intracellular processing and surface expression of an integral protein of the plasma membrane.

Abstract

We studied the effects of changes in microtubule assembly status upon the intracellular transport of an integral membrane protein from the rough endoplasmic reticulum to the plasma membrane. The protein was the G glycoprotein of vesicular stomatitis virus in cells infected with the Orsay-45 temperature-sensitive mutant of the virus; the synchronous intracellular transport of the G protein could be initiated by a temperature shift-down protocol. The intracellular and surface-expressed G protein were separately detected and localized in the same cells at different times after the temperature shift, by double-immunofluorescence microscopic measurements, and the extent of sialylation of the G protein at different times was quantitated by immunoprecipitation and SDS PAGE of [3SS]methionine-labeled cell extracts. Neither complete disassembly of the cytoplasmic microtubules by nocodazole treatment, nor the radical reorganization of microtubules upon taxol treatment, led to any perceptible changes in the rate or extent of G protein sialylation, nor to any marked changes in the rate or extent of surface appearance of the G protein. However, whereas in control cells the surface expression of G was polarized, at membrane regions in juxtaposition to the perinuclear compact Golgi apparatus, in cells with disassembled microtubules the surface expression of the G protein was uniform, corresponding to the intracellular dispersal of the elements of the Golgi apparatus. The mechanisms of transfer of integral proteins from the rough endoplasmic reticulum to the Golgi apparatus, and from the Golgi apparatus to the plasma membrane, are discussed in the light of these observations, and compared with earlier studies of the intracellular transport of secretory proteins.There is good evidence that, during the intracellular transport of secretory and membrane components, the transfer of these components from the Golgi apparatus (GA) ~ to the cell surface occurs by way of specific vesicles that bud off the trans face of the GA and migrate to the plasma membrane (for a recent review, see reference 13). It is thought that a similar process occurs to transfer these secretory and membrane components from their sites of synthesis and insertion in the rough endoplasmic reticulum (RER) to the GA, with so-called "transition Abbreviations used in this paper: GA, Golgi apparatus; NRK, normal rat kidney; 0-45, temperature-sensitive mutant of vesicular stomatitis virus Orsay-45; RER, rough and endoplasmic reticulum; VSV, vesicular stomatitis virus.

Microtubule-dependent control of cell shape and pseudopodial activity is inhibited by the antibody to kinesin motor domain.

“…Certain models of cell locomotion involve the addition of new membrane at the leading edge (for review see Singer and Kupfer, 1986); this edge was reported to be the primary site of microtubule-dependent incorporation of membrane marker protein (Bergmann et al, 1983;Rogalski et al, 1984). HD injections could probably stop this membrane addition and thus suppress pseudopodial activity and cell locomotion.…”

Differing polarity of the constitutive and regulated secretory pathways for von Willebrand factor in endothelial cells.

“…The situation that exists for membrane protein transport, however, appears to be different. The rate and extent of VSV G protein surface expression in virusinfected fibroblasts is not altered due to microtubular disruption (Rogalski et al, 1984), and conflicting reports of the effect of these agents on the polarity of viral protein transport exist. Rogalski et al (1984) suggested that the polarity of VSV G protein surface expression is altered because of microtubular disruption.…”

“…The rate and extent of VSV G protein surface expression in virusinfected fibroblasts is not altered due to microtubular disruption (Rogalski et al, 1984), and conflicting reports of the effect of these agents on the polarity of viral protein transport exist. Rogalski et al (1984) suggested that the polarity of VSV G protein surface expression is altered because of microtubular disruption. Rindler et al (1987) observed no alteration in polarity of VSV G protein surface expression with microtubular disruption, but did observe altered polarity of influenza hemagglutinin surface expression.…”

Characterization of a Novel Giant Scaffolding Protein, CG-NAP, That Anchors Multiple Signaling Enzymes to Centrosome and the Golgi Apparatus

“…4C, a and b). We further examined the relationship between CG-NAP and the Golgi apparatus using the microtubule-destabilizing agent nocodazole (27) and the fungal metabolite BFA (28), which are known to disrupt the Golgi apparatus by distinct mechanisms. Nocodazole treatment of HeLa cells dispersed the perinuclear CG-NAP staining into scattered pattern throughout the cytoplasm (Fig.…”