An Aspartic Endopeptidase is Involved in the Breakdown of Propeptides of Storage Proteins in Protein-Storage Vacuoles of Plants

Storage proteins are deposited into protein storage vacuoles (PSVs) during plant seed development and maturation and stably accumulate to high levels; subsequently, during germination the storage proteins are rapidly degraded to provide nutrients for use by the embryo. Here, we show that a PSV has within it a membrane-bound compartment containing crystals of phytic acid and proteins that are characteristic of a lytic vacuole. This compound organization, a vacuole within a vacuole whereby storage functions are separated from lytic functions, has not been described previously for organelles within the secretory pathway of eukaryotic cells. The partitioning of storage and lytic functions within the same vacuole may reflect the need to keep the functions separate during seed development and maturation and yet provide a ready source of digestive enzymes to initiate degradative processes early in germination.

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Structural insights into how vacuolar sorting receptors recognize the sorting determinants of seed storage proteins

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2111281119 ◽

2022 ◽

Vol 119 (1) ◽

pp. e2111281119

Author(s):

Hsi-En Tsao ◽

Shu Nga Lui ◽

Anthony Hiu-Fung Lo ◽

Shuai Chen ◽

Hiu Yan Wong ◽

...

Keyword(s):

Fluorescent Protein ◽

Storage Proteins ◽

Salt Bridges ◽

Protein Storage ◽

Protein Storage Vacuoles ◽

C Terminus ◽

Vacuolar Sorting ◽

Structural Insights ◽

Cargo Binding

In Arabidopsis, vacuolar sorting receptor isoform 1 (VSR1) sorts 12S globulins to the protein storage vacuoles during seed development. Vacuolar sorting is mediated by specific protein–protein interactions between VSR1 and the vacuolar sorting determinant located at the C terminus (ctVSD) on the cargo proteins. Here, we determined the crystal structure of the protease-associated domain of VSR1 (VSR1-PA) in complex with the C-terminal pentapeptide (468RVAAA472) of cruciferin 1, an isoform of 12S globulins. The 468RVA470 motif forms a parallel β-sheet with the switch III residues (127TMD129) of VSR1-PA, and the 471AA472 motif docks to a cradle formed by the cargo-binding loop (95RGDCYF100), making a hydrophobic interaction with Tyr99. The C-terminal carboxyl group of the ctVSD is recognized by forming salt bridges with Arg95. The C-terminal sequences of cruciferin 1 and vicilin-like storage protein 22 were sufficient to redirect the secretory red fluorescent protein (spRFP) to the vacuoles in Arabidopsis protoplasts. Adding a proline residue to the C terminus of the ctVSD and R95M substitution of VSR1 disrupted receptor–cargo interactions in vitro and led to increased secretion of spRFP in Arabidopsis protoplasts. How VSR1-PA recognizes ctVSDs of other storage proteins was modeled. The last three residues of ctVSD prefer hydrophobic residues because they form a hydrophobic cluster with Tyr99 of VSR1-PA. Due to charge–charge interactions, conserved acidic residues, Asp129 and Glu132, around the cargo-binding site should prefer basic residues over acidic ones in the ctVSD. The structural insights gained may be useful in targeting recombinant proteins to the protein storage vacuoles in seeds.

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Transport of storage proteins to the vacuole is mediated by vesicles without a clathrin coat

Journal of Cell Science ◽

10.1242/jcs.109.10.2539 ◽

1996 ◽

Vol 109 (10) ◽

pp. 2539-2550 ◽

Cited By ~ 1

Author(s):

I. Hohl ◽

D.G. Robinson ◽

M.J. Chrispeels ◽

G. Hinz

Keyword(s):

Protein Transport ◽

Storage Proteins ◽

Immunogold Labelling ◽

Acid Hydrolases ◽

Protein Storage ◽

Protein Storage Vacuoles ◽

Different Types ◽

Pass Through ◽

Vacuolar Protein ◽

Dense Vesicles

Storage parenchyma cells of developing legume cotyledons actively transport large amounts of storage proteins to protein storage vacuoles (PSV). These proteins are synthesized on the endoplasmic reticulum and pass through the Golgi apparatus. Clathrin coated vesicles (CCV) and small electron dense vesicles found near the trans-Golgi network (TGN) have both been implicated in the Golgi-to-vacuole transport step. Recent findings that protein storage cells contain more than one type of vacuole have necessitated a re-examination of the role of both types of vesicles in vacuolar protein transport. Immunoblots of highly purified CCV preparations and immunogold labelling with antibodies to the storage proteins vicilin and legumin, indicate that the dense vesicles, but not the CCV, are involved in storage protein transport in pea cotyledons. This result is supported by the finding that alpha-TIP, a protein characteristic of the PSV membrane, is absent from CCV. In addition, complex glycoproteins appear to be carried by CCV but are not detectable in the PSV. We suggest on the basis of these data that storage proteins and other vacuolar proteins such as acid hydrolases are not sorted by the same mechanism and are transported by different types of vesicles to different types of vacuoles.

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Protein storage vacuoles form de novo during pea cotyledon development

Journal of Cell Science ◽

10.1242/jcs.108.1.299 ◽

1995 ◽

Vol 108 (1) ◽

pp. 299-310 ◽

Cited By ~ 2

Author(s):

B. Hoh ◽

G. Hinz ◽

B.K. Jeong ◽

D.G. Robinson

Keyword(s):

Membrane Protein ◽

De Novo ◽

Storage Proteins ◽

Protein Body ◽

Membrane System ◽

Intermediate Stage ◽

Integral Membrane Protein ◽

Protein Storage ◽

Protein Storage Vacuoles ◽

Protein Storage Vacuole

We have investigated the formation of protein storage vacuoles in peas (Pisum sativum L.) in order to determine whether this organelle arises de novo during cotyledon development. A comparison of different stages in cotyledon development indicates that soluble protease activities decline and the amounts of storage proteins and the integral membrane protein of the protein body, alpha-TIP, increase during seed maturation. On linear sucrose density gradients we have been able to distinguish between two separate vesicle populations: one enriched in alpha-TIP, and one in TIP-Ma 27, a membrane protein characteristic of vegetative vacuoles. Both vesicle populations possess, however, PPase and V-ATPase activities. Conventionally fixed cotyledonary tissue at an intermediate stage in cotyledon development reveals the presence of a complex tubular-cisternal membrane system that seems to surround the pre-existing vacuoles. The latter gradually become compressed as a result of dilation of the former membrane system. This was confirmed immunocytochemically with the TIP-Ma 27 antiserum. Deposits of the storage proteins vicilin and legumin in the lumen, and the presence of alpha-TIP in the membranes of the expanding membrane system provide evidence of its identity as a precursor to the protein storage vacuole.

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The small GTPase Rab5a and its guanine nucleotide exchange factors are involved in post-Golgi trafficking of storage proteins in developing soybean cotyledon

Journal of Experimental Botany ◽

10.1093/jxb/erz454 ◽

2019 ◽

Vol 71 (3) ◽

pp. 808-822 ◽

Cited By ~ 1

Author(s):

Zhongyan Wei ◽

Tian Pan ◽

Yuyang Zhao ◽

Bohong Su ◽

Yulong Ren ◽

...

Keyword(s):

Protein Trafficking ◽

Storage Protein ◽

Storage Proteins ◽

Small Gtpase ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Protein Storage ◽

Soybean Cotyledon ◽

Guanine Nucleotide Exchange ◽

Protein Storage Vacuoles

Abstract Storage protein is the most abundant nutritional component in soybean seed. Morphology-based evidence has verified that storage proteins are initially synthesized on the endoplasmic reticulum, and then follow the Golgi-mediated pathway to the protein storage vacuole. However, the molecular mechanisms of storage protein trafficking in soybean remain unknown. Here, we clone the soybean homologs of Rab5 and its guanine nucleotide exchange factor (GEF) VPS9. GEF activity combined with yeast two-hybrid assays demonstrated that GmVPS9a2 might specifically act as the GEF of the canonical Rab5, while GmVPS9b functions as a common activator for all Rab5s. Subcellular localization experiments showed that GmRab5a was dually localized to the trans-Golgi network and pre-vacuolar compartments in developing soybean cotyledon cells. Expression of a dominant negative variant of Rab5a, or RNAi of either Rab5a or GmVPS9s, significantly disrupted trafficking of mRFP–CT10, a cargo marker for storage protein sorting, to protein storage vacuoles in maturing soybean cotyledons. Together, our results systematically revealed the important role of GmRab5a and its GEFs in storage protein trafficking, and verified the transient expression system as an efficient approach for elucidating storage protein trafficking mechanisms in seed.

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Self-aggregation of legume seed storage proteins inside the protein storage vacuoles is electrostatic in nature, rather than lectin-mediated

FEBS Letters ◽

10.1016/s0014-5793(02)03801-2 ◽

2002 ◽

Vol 534 (1-3) ◽

pp. 106-110 ◽

Cited By ~ 11

Author(s):

Ricardo B. Ferreira ◽

Regina L. Freitas ◽

Artur R. Teixeira

Keyword(s):

Storage Proteins ◽

Seed Storage ◽

Seed Storage Proteins ◽

Legume Seed ◽

Protein Storage ◽

Protein Storage Vacuoles ◽

Self Aggregation

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Mass Transport of Proform of a Kdel-Tailed Cysteine Proteinase (Sh-EP) to Protein Storage Vacuoles by Endoplasmic Reticulum–Derived Vesicle Is Involved in Protein Mobilization in Germinating Seeds

The Journal of Cell Biology ◽

10.1083/jcb.148.3.453 ◽

2000 ◽

Vol 148 (3) ◽

pp. 453-464 ◽

Cited By ~ 117

Author(s):

Kiminori Toyooka ◽

Takashi Okamoto ◽

Takao Minamikawa

Keyword(s):

Endoplasmic Reticulum ◽

Mass Transport ◽

Intracellular Transport ◽

Storage Proteins ◽

Cysteine Proteinase ◽

Transport Pathway ◽

Protein Storage ◽

Protein Storage Vacuoles ◽

Protein Mobilization ◽

Cotyledon Cells

A vacuolar cysteine proteinase, designated SH-EP, is expressed in the cotyledon of germinated Vigna mungo seeds and is responsible for the degradation of storage proteins. SH-EP is a characteristic vacuolar proteinase possessing a COOH-terminal endoplasmic reticulum (ER) retention sequence, KDEL. In this work, immunocytochemical analysis of the cotyledon cells of germinated V. mungo seeds was performed using seven kinds of antibodies to identify the intracellular transport pathway of SH-EP from ER to protein storage vacuoles. A proform of SH-EP synthesized in ER accumulated at the edge or middle region of ER where the transport vesicle was formed. The vesicle containing a large amount of proSH-EP, termed KV, budded off from ER, bypassed the Golgi complex, and was sorted to protein storage vacuoles. This massive transport of SH-EP via KV was thought to mediate dynamic protein mobilization in the cotyledon cells of germinated seeds. We discuss the possibilities that the KDEL sequence of KDEL-tailed vacuolar cysteine proteinases function as an accumulation signal at ER, and that the mass transport of the proteinases by ER-derived KV-like vesicle is involved in the protein mobilization of plants.

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Comparative cytological and biochemical analysis of protein storage vacuoles from cotyledons and radicles of cotton seeds

Seed Science Research ◽

10.1017/s096025850000297x ◽

1996 ◽

Vol 6 (1) ◽

pp. 31-37 ◽

Cited By ~ 1

Author(s):

E.L. Vigil ◽

A.L. Fleming ◽

T. Fang ◽

N. Chaney ◽

W. P. Wergin

Keyword(s):

Mineral Composition ◽

Storage Proteins ◽

Radicle Growth ◽

Protein Storage ◽

Protein Storage Vacuoles ◽

Sds Page ◽

Lower Molecular Mass ◽

Elemental Analyses ◽

Cotton Seeds ◽

Protein Complement

AbstractProtein storage vacuoles (PSVs) from radicles and cotyledons of dry cotton seeds were isolated by differential centrifugation following homogenization in glycerol. Protein complement analysis of isolated PSVs with one dimensional SDS-PAGE gels revealed similar major storage proteins, viz. 53 and 48 kDa, with differences in lower molecular mass proteins. Radicle PSVs have apparently more 35-kDa and less 22-kDa storage protein than do cotyledon PSVs. The mineral composition of whole radicles, cotyledons and isolated PSVs from radicles and cotyledons was determined by atomic absorption spectroscopy and colorimetric elemental analyses. The concentration of calcium (Ca), magnesium (Mg), potassium (K) and phosphate (P) was lower in isolated PSVs from radicles than from cotyledons, resulting in a marked difference in the Mg/Ca and (Mg+Ca)/K ratios in PSVs from these two sources. Analysis of radicle and cotyledon tissue from dry seeds for mineral distribution with EDX and scanning electron microscopy revealed major concentrations of Mg, K and P in PSVs. These observations indicate that PSVs in radicles are similar in protein and mineral composition to PSVs in cotyledons. PSVs in radicles have the potential function as storage organelles to provide minerals and nutrients for radicle growth during imbibition and germination.

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