Storage Protein Accumulation in the Absence of the Vacuolar Processing Enzyme Family of Cysteine Proteases[W]

Abstract

The role(s) of specific proteases in seed protein processing is only vaguely understood; indeed, the overall role of processing in stable protein deposition has been the subject of more speculation than direct investigation. Seed-type members of the vacuolar processing enzyme (VPE) family were hypothesized to perform a unique function in seed protein processing, but we demonstrated previously that Asn-specific protein processing in developing Arabidopsis seeds occurs independently of this VPE activity. Here, we describe the unexpected expression of vegetative-type VPEs in developing seeds and test the role(s) of all VPEs in seed storage protein accumulation by systematically stacking knockout mutant alleles of all four members ( ␣ VPE , ␤ VPE , ␥ VPE , and ␦ VPE ) of the VPE gene family in Arabidopsis. The complete removal of VPE function in the ␣ vpe ␤ vpe ␥ vpe ␦ vpe quadruple mutant resulted in a total shift of storage protein accumulation from wild-type processed polypeptides to a finite number of prominent alternatively processed polypeptides cleaved at sites other than the conserved Asn residues targeted by VPE. Although alternatively proteolyzed legumin-type globulin polypeptides largely accumulated as intrasubunit disulfide-linked polypeptides with apparent molecular masses similar to those of VPE-processed legumin polypeptides, they showed markedly altered solubility and protein assembly characteristics. Instead of forming 11S hexamers, alternatively processed legumin polypeptides were deposited primarily as 9S complexes. However, despite the impact on seed protein processing, plants devoid of all known functional VPE genes appeared unchanged with regard to protein content in mature seeds, relative mobilization rates of protein reserves during germination, and vegetative growth. These findings indicate that VPE-mediated Asn-specific proteolytic processing, and the physiochemical property changes attributed to this specific processing step, are not required for the successful deposition and mobilization of seed storage protein in the protein storage vacuoles of Arabidopsis seeds.

A Pivotal Role of the Basic Leucine Zipper Transcription Factor bZIP53 in the Regulation ofArabidopsisSeed Maturation Gene Expression Based on Heterodimerization and Protein Complex Formation

“…However, as observed before, the presence of MAT RNA does not necessarily result in the detection of the corresponding proteins. Additional cellular requirements and tissue-specific protein degradation determine high-level accumulation of MAT proteins (Gruis et al, 2004) Complementing the gain-of-function results, reduced expression of bZIP53 in a knockdown line that harbors a T-DNA insertion in the promoter leads to strong reduction in the expression of the corresponding target genes. However, these plants still set viable seeds, which might be due to a residual amount of bZIP53 protein.…”

The Proteolytic Processing of Seed Storage Proteins inArabidopsisEmbryo Cells Starts in the Multivesicular Bodies

“…The Arabidopsis 2S albumins are exported from the ER as precursors that contain three propeptides (an N-terminal propeptide, an internal propeptide, and a C-terminal propeptide). These propeptides are removed posttranslationally by proteolytic processing enzymes (Gruis et al, 2002(Gruis et al, , 2004Shimada et al, 2003b).…”

Seed storage albumins: biosynthesis, trafficking and structures

“…Quadruple aep null plants had a dramatically different seed protein profile, but this defective processing did not affect seed germination or the adult plant phenotype, which makes one wonder what the purpose of all this post-translational processing is? A close analysis of the proteins in aep null seeds showed that in the absence of AEP, rather than remain completely unprocessed the proalbumins (and proglobulins) were processed improperly and inaccurately by other proteases that otherwise played little or no role in albumin maturation (Shimada et al 2003b;Gruis et al 2004). Proteomic analysis of the albumin in Arabidopsis wild type and aep null plants also showed that one of the four Arabidopsis SEED STORAGE ALBUMIN (SESA) proteins was not fully dependent upon AEP for its maturation.…”

“…Arabidopsis aep2 mutants had mild defects in seed storage protein profile (Gruis et al 2002;Shimada et al 2003b). Single mutation in the other three AEP genes did not give noticeable changes in seed protein profile, but when lesions in AEP genes were pyramided the resulting plants had dramatically perturbed seed storage protein profiles (Shimada et al 2003b;Gruis et al 2004). Quadruple aep null plants had a dramatically different seed protein profile, but this defective processing did not affect seed germination or the adult plant phenotype, which makes one wonder what the purpose of all this post-translational processing is?…”

“…Proteomic analysis of the albumin in Arabidopsis wild type and aep null plants also showed that one of the four Arabidopsis SEED STORAGE ALBUMIN (SESA) proteins was not fully dependent upon AEP for its maturation. Specifically the large albumin subunit of SESA3 (At4g27160, At2S-3) is N-terminally matured in an AEP-independent fashion although by which protease is not clear (Shimada et al 2003b;Gruis et al 2004).…”