Clathrin-coated vesicles contain an ATP-dependent proton pump

Area of Science:

• Cell Biology
• Biochemistry
• Membrane Transport

Background:

• Clathrin-coated vesicles are crucial for intracellular trafficking.
• The internal pH of vesicles influences various cellular processes, including endocytosis.

Purpose of the Study:

• To investigate the presence and function of a proton pump in clathrin-coated vesicles.
• To determine the bioenergetics and characteristics of this proton pump.

Main Methods:

• Isolation of clathrin-coated vesicles from calf brain.
• Measurement of proton movement using [14C]methylamine distribution.
• Assay of ATPase activity in the presence of various inhibitors and ionophores.

Main Results:

• An ATP-dependent proton pump was identified, generating a significant pH gradient (0.6-0.7 units).
• Proton uptake was sensitive to protonophores (FCCP) and carboxyl reagents but not to Na+, K+-ATPase or mitochondrial ATPase inhibitors.
• GTP supported proton uptake, while a non-hydrolyzable ATP analog did not.
• Membrane potential dissipation stimulated proton uptake, and FCCP/valinomycin stimulated ATPase activity.

Conclusions:

• Clathrin-coated vesicles contain an electrogenic, ATP-dependent proton pump.
• This proton pump likely contributes to the acidification required for receptor-mediated endocytosis.