Trafficking of Vacuolar Proteins: The Crucial Role of Arabidopsis Vacuolar Protein Sorting 29 in Recycling Vacuolar Sorting Receptor

The retromer is involved in recycling lysosomal sorting receptors in mammals. A component of the retromer complex in Arabidopsis thaliana, vacuolar protein sorting 29 (VPS29), plays a crucial role in trafficking storage proteins to protein storage vacuoles. However, it is not known whether or how vacuolar sorting receptors (VSRs) are recycled from the prevacuolar compartment (PVC) to the trans-Golgi network (TGN) during trafficking to the lytic vacuole (LV). Here, we report that VPS29 plays an essential role in the trafficking of soluble proteins to the LV from the TGN to the PVC. maigo1-1 (mag1-1) mutants, which harbor a knockdown mutation in VPS29, were defective in trafficking of two soluble proteins, Arabidopsis aleurain-like protein (AALP):green fluorescent protein (GFP) and sporamin:GFP, to the LV but not in trafficking membrane proteins to the LV or plasma membrane or via the secretory pathway. AALP:GFP and sporamin:GFP in mag1-1 protoplasts accumulated in the TGN but were also secreted into the medium. In mag1-1 mutants, VSR1 failed to recycle from the PVC to the TGN; rather, a significant proportion was transported to the LV; VSR1 overexpression rescued this defect. Moreover, endogenous VSRs were expressed at higher levels in mag1-1 plants. Based on these results, we propose that VPS29 plays a crucial role in recycling VSRs from the PVC to the TGN during the trafficking of soluble proteins to the LV.     

MAIGO2 is involved in exit of seed storage proteins from the endoplasmic reticulum in Arabidopsis thaliana

Seed storage proteins are synthesized on the endoplasmic reticulum (ER) as precursors and then transported to protein storage vacuoles, where they are processed into mature forms. Here, we isolated an Arabidopsis thaliana mutant, maigo2 (mag2), that accumulated the precursors of two major storage proteins, 2S albumin and 12S globulin, in dry seeds. mag2 seed cells contained many novel structures, with an electron-dense core that was composed of the precursor forms of 2S albumin. 12S globulins were segregated from 2S albumin and were localized in the matrix region of the structures together with the ER chaperones lumenal binding protein and protein disulfide isomerase, which were more abundant in mag2 seeds. The MAG2 gene was identified as At3g47700, and the MAG2 protein had a RINT-1/TIP20 domain in the C-terminal region. We found that some MAG2 molecules were peripherally associated with the ER membrane. MAG2 had an ability to bind to two ER-localized t-SNAREs (for target-soluble NSF [N-ethylmaleimide-sensitive fusion protein] attachment protein receptor; At Sec20 and At Ufe1). Our findings suggest that MAG2 functions in the transport of storage protein precursors between the ER and Golgi complex in plants.     

Characterization of histidinol phosphate aminotransferase from Escherichia coli

Histidinol phosphate aminotransferase (HPAT) is a pyridoxal 5'-phosphate (PLP)-dependent aminotransferase classified into Subgroup I aminotransferase, in which aspartate aminotransferase (AspAT) is the prototype. In order to expand our knowledge on the reaction mechanism of Subgroup I aminotransferases, HPAT is an enzyme suitable for detailed mechanistic studies because of having low sequence identity with AspAT and a unique substrate recognition mode. Here we investigated the spectroscopic properties of HPAT and the effect of the C4-C4' strain of the PLP-Lys(214) Schiff base on regulating the Schiff base pK(a) in HPAT. Similar to AspAT, the PLP-form HPAT showed pH-dependent absorption spectral change with maxima at 340 nm at high pH and 420 nm at low pH, having a low pK(a) of 6.6. The pK(a) value of the methylamine-reconstituted K214A mutant enzyme was increased from 6.6 to 10.6. Mutation of Asn(157) to Ala increased the pK(a) to 9.2. Replacement of Arg(335) by Leu increased the pK(a) to 8.6. On the other hand, the pK(a) value of the N157A/R335L double mutant enzyme was 10.6. These data indicate that the strain of the Schiff base is the principal factor to decrease the pK(a) in HPAT and is crucial for the subsequent increase in the Schiff base pK(a) during catalysis, although the electrostatic effect of the arginine residue that binds the negatively charged group of the substrate is larger in HPAT than that in AspAT. Our findings also support the idea that the strain mechanism is common to Subgroup I aminotransferases.     

Domain structure of chondroitin sulfate E octasaccharides binding to type V collagen.

We demonstrated previously that chondroitin sulfate E (ChS-E) binds to type V collagen (Munakata, H., Takagaki, K., Majima, M., and Endo, M. (1999) Glycobiology 9, 1023--1027). In this study, we investigated the structure and binding of ChS-E oligosaccharides. Eleven oligosaccharides were isolated from ChS-E by gel filtration chromatography and anion-exchange high performance liquid chromatography after hydrolysis with testicular hyaluronidase. Separately, seven oligosaccharides were custom synthesized using the transglycosylation reaction of testicular hyaluronidase. Structural analysis was performed by enzymatic digestions in conjunction with high performance liquid chromatography and mass spectrometry. This library of 18 oligosaccharides was used as a source of model molecules to clarify the structural requirements for binding to type V collagen. Binding was analyzed by a biosensor based on surface plasmon resonance. The results indicated that to bind to type V collagen the oligosaccharides must have the following carbohydrate structures: 1) octasaccharide or larger in size; 2) a continuous sequence of three GlcAbeta1--3GalNAc(4S,6S) units; 3) a GlcAbeta1--3GalNAc(4S,6S) unit, GlcAbeta1--3GalNAc(4S) unit or GlcAbeta1--3GalNAc(6S) unit at the reducing terminal; 4) a GlcAbeta1--3GalNAc(4S,6S) unit at the nonreducing terminal. It is likely that these characteristic oligosaccharide sequences play key roles in cell adhesion and extracellular matrix assembly.     

SCRAPPER-dependent ubiquitination of active zone protein RIM1 regulates synaptic vesicle release

Little is known about how synaptic activity is modulated in the central nervous system. We have identified SCRAPPER, a synapse-localized E3 ubiquitin ligase, which regulates neural transmission. SCRAPPER directly binds and ubiquitinates RIM1, a modulator of presynaptic plasticity. In neurons from Scrapper-knockout (SCR-KO) mice, RIM1 had a longer half-life with significant reduction in ubiquitination, indicating that SCRAPPER is the predominant ubiquitin ligase that mediates RIM1 degradation. As anticipated in a RIM1 degradation defect mutant, SCR-KO mice displayed altered electrophysiological synaptic activity, i.e., increased frequency of miniature excitatory postsynaptic currents. This phenotype of SCR-KO mice was phenocopied by RIM1 overexpression and could be rescued by re-expression of SCRAPPER or knockdown of RIM1. The acute effects of proteasome inhibitors, such as upregulation of RIM1 and the release probability, were blocked by the impairment of SCRAPPER. Thus, SCRAPPER has an essential function in regulating proteasome-mediated degradation of RIM1 required for synaptic tuning.     

Biosynthetic origin of the carbon skeleton and nitrogen atom of pamamycin-607, a nitrogen-containing polyketide

The biosynthesis of pamamycin-607 (PM-607), a sixteen-membered macrodiolide compound, was studied with 13C- and 15N-labeled precursor units in Streptomyces alboniger. Feeding experiments with 13C-labeled acetate or propionate indicate that the carbon skeleton of PM-607 was derived from six acetate, four propionate and three succinate units. MS analyses of 15N-labeled PM-607 suggest that the nitrogen atom in PM-607 was derived from the alpha-amino group of an amino acid.     

An asparaginyl endopeptidase mediates in vivo protein backbone cyclization

Proteases can catalyze both peptide bond cleavage and formation, yet the hydrolysis reaction dominates in nature. This presents an interesting challenge for the biosynthesis of backbone cyclized (circular) proteins, which are encoded as part of precursor proteins and require post-translational peptide bond formation to reach their mature form. The largest family of circular proteins are the plant-produced cyclotides; extremely stable proteins with applications as bioengineering scaffolds. Little is known about the mechanism by which they are cyclized in vivo but a highly conserved Asn (occasionally Asp) residue at the C terminus of the cyclotide domain suggests that an enzyme with specificity for Asn (asparaginyl endopeptidase; AEP) is involved in the process. Nicotiana benthamiana does not endogenously produce circular proteins but when cDNA encoding the precursor of the cyclotide kalata B1 was transiently expressed in the plants they produced the cyclotide, together with linear forms not commonly observed in cyclotide-containing plants. Observation of these species over time showed that in vivo asparaginyl bond hydrolysis is necessary for cyclization. When AEP activity was suppressed, either by decreasing AEP gene expression or using a specific inhibitor, the amount of cyclic cyclotide in the plants was reduced compared with controls and was accompanied by the accumulation of extended linear species. These results suggest that an AEP is responsible for catalyzing both peptide bond cleavage and ligation of cyclotides in a single processing event.     

Legumain/asparaginyl endopeptidase controls extracellular matrix remodeling through the degradation of fibronectin in mouse renal proximal tubular cells

Legumain/asparaginyl endopeptidase (EC 3.4.22.34) is a novel cysteine protease that is abundantly expressed in the late endosomes and lysosomes of renal proximal tubular cells. Recently, emerging evidence has indicated that legumain might play an important role in control of extracellular matrix turnover in various pathological conditions such as tumor growth/metastasis and progression of atherosclerosis. We initially found that purified legumain can directly degrade fibronectin, one of the main components of the extracellular matrix, in vitro. Therefore, we examined the effect of legumain on fibronectin degradation in cultured mouse renal proximal tubular cells. Fibronectin processing can be inhibited by chloroquine, an inhibitor of lysosomal degradation, and can be enhanced by the overexpression of legumain, indicating that fibronectin degradation occurs in the presence of legumain in lysosomes from renal proximal tubular cells. Furthermore, in legumain-deficient mice, unilateral ureteral obstruction (UUO)-induced renal interstitial protein accumulation of fibronectin and renal interstitial fibrosis were markedly enhanced. These findings indicate that legumain might have an important role in extracellular matrix remodeling via the degradation of fibronectin in renal proximal tubular cells.