Activation of the Proteasomes of Sand Dollar Eggs at Fertilization Depends on the Intracellular pH Rise

Abstract

The mechanism of the activation of intracellular proteasomes at fertilization was measured in living sand dollar eggs using the membrane-impermeant fluorogenic substrate, succinyl-Phe-Leu-Arg-coumarylamido-4-methanesulfonic acid. When the substrate was microinjected into unfertilized eggs, the initial velocity of hydrolysis of the substrate (V0) was low.V0measured 5 to 10 min after fertilization was five to nine times the prefertilization level and remained high throughout the first cell cycle. Hydrolysis of the substrate was inhibited byclasto-lactacystin β-lactone, a specific inhibitor of the proteasome. There has beenin vitroevidence that calcium may be involved in regulation of proteasome activity to either inhibit the increase in peptidase activity associated with PA 28 binding to the 20S proteasome or stimulate activity of the PA 700–proteasome complex. Since both intracellular free Ca2+concentration ([Ca2+]i) and intracellular pH (pHi) increase after fertilization, hydrolysis of the proteasome substrate was measured under conditions in which [Ca2+]iand pHiwere varied independently during activation. When the pHiof unfertilized eggs was elevated by exposure to 15 mM ammonium chloride in pH 9 seawater,V0increased to a level comparable to that measured after fertilization. In contrast, [Ca2+]ielevation without pHichange, induced by calcium ionophore in sodium-free seawater, had no effect onV0in the unfertilized egg. Moreover, when unfertilized eggs were microinjected with buffers modulating pHi,V0increased in a pH-dependent manner. These results indicate that the pHirise at fertilization is the necessary prerequisite for activation of the proteasome, an essential component in the regulation of the cell cycle.