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

Area of Science:

• Cell Biology
• Molecular Biology
• Virology

Background:

• Intracellular transport is crucial for protein localization and function.
• Microtubules play a role in organelle positioning and vesicle trafficking.
• The G glycoprotein of vesicular stomatitis virus is a model for studying membrane protein transport.

Purpose of the Study:

• To investigate the role of microtubules in the intracellular transport of the G glycoprotein.
• To determine if microtubule integrity affects the rate or sialylation of G glycoprotein.
• To examine the impact of microtubule disruption on the polarized surface expression of G glycoprotein.

Main Methods:

• Utilized a temperature-sensitive mutant of vesicular stomatitis virus (Orsay-45).
• Employed temperature shift-down protocols to synchronize G glycoprotein transport.
• Applied nocodazole and taxol treatments to alter microtubule assembly.
• Used double-immunofluorescence microscopy for protein localization.
• Quantitated sialylation via immunoprecipitation and SDS-PAGE of [35S]methionine-labeled cell extracts.

Main Results:

• Neither microtubule disassembly nor reorganization affected the rate or extent of G glycoprotein sialylation.
• Microtubule disruption did not alter the rate or extent of G glycoprotein surface appearance.
• In control cells, G glycoprotein surface expression was polarized near the Golgi apparatus.
• In cells with disassembled microtubules, G glycoprotein surface expression was uniform due to Golgi dispersal.

Conclusions:

• Microtubules are not essential for the transport rate of G glycoprotein from the endoplasmic reticulum to the plasma membrane.
• Microtubule integrity is critical for the polarized localization of G glycoprotein at the cell surface.
• The Golgi apparatus's positioning, influenced by microtubules, dictates the polarized distribution of membrane proteins.