Inhibition of proteastome activity strongly affects kiwifruit pollen germination. Involvement of the ubiquitin/proteasome pathway as a major regulator

The 26S proteasome is a multicatalytic complex that acts as primary proteas e of the ubiquitin mediated proteolytic pathway in eukaryotes. We provide h ere the first evidence that the proteasome plays a key role in regulating p ollen tube growth. Immunoblotting experiments revealed the presence of high levels of free ubiquitin and ubiquitin conjugates in rehydrated and germin ating pollen of kiwifruit [Actinidia deliciosa var. deliciosa (A. Chev) C. F. Liang et A. R. Ferguson]. Proteasome activity, assayed fluorometrically, accompanied the progression of germination. Specific inhibitors of proteas ome function such as benzyloxycarbonyl-leucinyl-leucinyl-leucinal (MG-132), clasto-lactacystin beta -lactone, and epoxomicin significantly decreased t ube growth or altered tube morphology. High-molecular mass, ubiquitinated p roteins accumulated in MG-132- and beta -lactone-treated pollen, indicating that proteasome function was effectively impaired. The inhibitors were als o able to decrease in vitro proteasome activity in pollen extracts. Because MG-132 can inhibit calpains, as well as the proteasome, trans-epoxy succin yl-L-leucylamido-(4-guanidino) butane (E-64), an inhibitor of cysteine prot eases, was investigated. Some reduction in tube growth rare was observed, b ut only at 80 muM E-64, and no abnormal tubes were produced. Furthermore no inhibition of tube growth was observed when another inhibitor of cysteine proteases, leupeptin, or inhibitors of serine, and aspartic proteases (phen ylmethylsulfonyl fluoride and pepstatin) were used. Our results indicate th at protein turnover during tube organization and elongation in kiwifruit po llen is important, and our results also implicate the ubiquitin/26S proteas ome as the major proteolytic pathway involved.