Inhibition of Proteasome Activity Strongly Affects Kiwifruit Pollen Germination. Involvement of the Ubiquitin/Proteasome Pathway as a Major Regulator

Abstract

The 26S proteasome is a multicatalytic complex that acts as primary protease of the ubiquitin-mediated proteolytic pathway in eukaryotes. We provide here the first evidence that the proteasome plays a key role in regulating pollen tube growth. Immunoblotting experiments revealed the presence of high levels of free ubiquitin and ubiquitin conjugates in rehydrated and germinating 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 proteasome function such as benzyloxycarbonyl-leucinyl-leucinyl-leucinal (MG-132), clasto-lactacystin ␤-lactone, and epoxomicin significantly decreased tube growth or altered tube morphology. High-molecular mass, ubiquitinated proteins accumulated in MG-132-and ␤-lactone-treated pollen, indicating that proteasome function was effectively impaired. The inhibitors were also able to decrease in vitro proteasome activity in pollen extracts. Because MG-132 can inhibit calpains, as well as the proteasome, trans-epoxy succinyl-l-leucylamido-(4-guanidino) butane (E-64), an inhibitor of cysteine proteases, was investigated. Some reduction in tube growth rate was observed, but only at 80 m 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 (phenylmethylsulfonyl fluoride and pepstatin) were used. Our results indicate that protein turnover during tube organization and elongation in kiwifruit pollen is important, and our results also implicate the ubiquitin/26S proteasome as the major proteolytic pathway involved.

Cytoskeleton-Associated Large RNP Complexes in Tobacco Male Gametophyte (EPPs) Are Associated with Ribosomes and Are Involved in Protein Synthesis, Processing, and Localization

“…50 The importance of the selective proteolytic activity associated with 26S proteasome system during pollen germination and tube growth was repeatedly demonstrated. [51][52][53] Moreover, a number of transcripts encoding proteasome-associated and F-box proteins were identified in Arabidopsis developing and germinating pollen. 42,54 Proteasomes were also demonstrated to perform other nonproteolytic functions including transcriptional regulation, [55][56][57] RNase activity, 58,59 translational regulation, 60,61 and possessing cytoskeleton-binding properties.…”

The RPN1 Subunit of the 26S Proteasome in Arabidopsis Is Essential for Embryogenesis

“…Polyubiquinated proteins were analyzed in rpn1a/RPN1a plants, where homozygous mutant embryos may fail to degrade ubiquinated substrates, and in rpn1b/rpn1b homozygotes ( Figure 4C). If proteasome function were impaired in the mutants, this should affect the level of polyubiquitinated proteins, as it was described, for instance, after application of 26S proteasome inhibitors, which is accompanied by a drastic elevation in the general level of polyubiquitinated proteins (Speranza et al, 2001). Although the rpn1a mutant cells only constitute a small fraction of the cells from which protein extracts were made, a failure to degrade highly unstable substrates is expected to lead to changes in the profile of polyubiquitinated proteins (e.g., the appearance of new bands).…”

Proteomics Identification of Differentially Expressed Proteins Associated with Pollen Germination and Tube Growth Reveals Characteristics of Germinated Oryza sativa Pollen

“…26 S proteasome-based selective protein degradation is an important mechanism for temporal regulation and involves numerous cellular and development processes, including pollenstigma interaction (4) and polarized cell morphogenesis (55). The disruption of pollen germination by inhibitors of 26 S proteasome activity (56) and presynthesis of multiple subunit components of the complex in mature rice pollen (14) suggest that the pathway is implicated in regulation of pollen function. Furthermore our differential proteomics analysis identified six of 14 subunits of the 20 S proteolytic particle of the proteasome, all of which were up-regulated in growing tubes (Table I) as were some peptidases.…”