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

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.

scholarly journals Viable Polioviruses That Encode 2A Proteins with Fluorescent Protein Tags

2009 ◽  
Vol 84 (3) ◽  
pp. 1477-1488 ◽  
Author(s):  
Natalya L. Teterina ◽  
Eric A. Levenson ◽  
Ellie Ehrenfeld
Keyword(s):  
Fusion Protein ◽  
Fluorescent Protein ◽  
Viral Rna ◽  
Progeny Virus ◽  
C Terminus ◽  
Membrane Reorganization ◽  
Infected Cells ◽  
Viral Polyprotein ◽  
Fluorescent Protein Tags

ABSTRACT The 2A proteins of the Picornaviridae enterovirus genus are small cysteine proteinases that catalyze essential cleavages in the viral polyprotein in cis and in several cellular proteins in trans. In addition, 2A has been implicated in the process of viral RNA replication, independent of its protease functions. We have generated viable polioviruses that encode 2A proteins containing fluorescent protein tag insertions at either of two sites in the 2A protein structure. Viruses containing an insertion of Discosoma sp. red fluorescent protein (DsRed) after residue 144 of 2A, near the C terminus, produced plaques only slightly smaller than wild-type (wt) virus. The polyprotein harboring the 2A-DsRed fusion protein was efficiently and accurately cleaved; fluorescent 2A proteinase retained protease activity in trans and supported translation and replication of viral RNA, both in vitro and in infected cells. Intracellular membrane reorganization to support viral RNA synthesis was indistinguishable from that induced by wt virus. Infected cells exhibited strong red fluorescence from expression of the 2A-DsRed fusion protein, and the progeny virus was stable for three to four passages, after which deletions within the DsRed coding sequence began to accumulate. Confocal microscopic imaging and analysis revealed a portion of 2A-DsRed in punctate foci concentrated in the perinuclear region that colocalized with replication protein 2C. The majority of 2A, however, was associated with an extensive structural matrix throughout the cytoplasm and was not released from infected cells permeabilized with digitonin.

Missing pieces in protein deposition and mobilization inside legume seed storage vacuoles: calcium and magnesium ions

2012 ◽  
Vol 22 (4) ◽  
pp. 249-258 ◽  
Author(s):  
Cláudia N. Santos ◽  
Marta M. Alves ◽  
Isabel T. Bento ◽  
Ricardo B. Ferreira
Keyword(s):  
Alkaline Earth ◽  
Storage Proteins ◽  
Lupinus Albus ◽  
Physiological Role ◽  
Experimental Conditions ◽  
Protein Storage Vacuoles ◽  
Aggregation And Disaggregation ◽  
Alkaline Earth Cations

AbstractDuring the maturation of dicotyledonous seeds, organic carbon, nitrogen and sulphur are stored in protein storage vacuoles (PSVs) as storage globulins. Several studies point to the coexistence of storage proteins with proteases responsible for their degradation inside PSVs. Different mechanisms have been proposed to explain why there is no proteolysis during this period. Protein aggregation to form large supramolecular structures resistant to proteolytic attack could be the reason. However, during germination, and particularly following its completion, the globulin aggregates must undergo disintegration to allow protease attack for protein reserve mobilization. Based on the well-described concentration-dependent ability of Ca2+ and Mg2+ to promote in vitro aggregation and disaggregation of globulins, we explored a possible role for these alkaline earth cations in globulin packaging and mobilization. Ca2+ and Mg2+ measurements in purified PSVs [6.37 μmol and 43.9 μmol g− 1 dry weight (DW) of cotyledons, respectively] showed the presence of these two alkaline earth cations within this compartment. To our knowledge, this is the first time that Ca2+ and Mg2+ have been quantified in purified PSVs from Lupinus albus seeds. Considering the importance of these two alkaline earth cations inside PSVs, which represent 14.6% and 60.7% of the total seed Mg2+and Ca2+, respectively, globulin aggregation and disaggregation profiles were assayed using experimental conditions closer to those that are physiologically present (proportion of Ca2+ and Mg2+, and acidic pH). Based on: (1) the high in vivo abundance of Ca2+ and Mg2+ inside PSVs; and (2) globulin aggregation and disaggregation profiles, together with structural and physiological evidence already reported in the literature, an important physiological role for Ca2+ and Mg2+ in globulin packaging and mobilization inside PSVs is suggested.

scholarly journals Interaction of the Endocytic Scaffold Protein Pan1 with the Type I Myosins Contributes to the Late Stages of Endocytosis

2007 ◽  
Vol 18 (8) ◽  
pp. 2893-2903 ◽  
Author(s):  
Sarah L. Barker ◽  
Linda Lee ◽  
B. Daniel Pierce ◽  
Lymarie Maldonado-Báez ◽  
David G. Drubin ◽  
...  
Keyword(s):  
Late Stage ◽  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Temperature Sensitive ◽  
Type I ◽  
Reading Frame ◽  
Rich Domain ◽  
C Terminus

The yeast endocytic scaffold Pan1 contains an uncharacterized proline-rich domain (PRD) at its carboxy (C)-terminus. We report that the pan1-20 temperature-sensitive allele has a disrupted PRD due to a frame-shift mutation in the open reading frame of the domain. To reveal redundantly masked functions of the PRD, synthetic genetic array screens with a pan1ΔPRD strain found genetic interactions with alleles of ACT1, LAS17 and a deletion of SLA1. Through a yeast two-hybrid screen, the Src homology 3 domains of the type I myosins, Myo3 and Myo5, were identified as binding partners for the C-terminus of Pan1. In vitro and in vivo assays validated this interaction. The relative timing of recruitment of Pan1-green fluorescent protein (GFP) and Myo3/5-red fluorescent protein (RFP) at nascent endocytic sites was revealed by two-color real-time fluorescence microscopy; the type I myosins join Pan1 at cortical patches at a late stage of internalization, preceding the inward movement of Pan1 and its disassembly. In cells lacking the Pan1 PRD, we observed an increased lifetime of Myo5-GFP at the cortex. Finally, Pan1 PRD enhanced the actin polymerization activity of Myo5–Vrp1 complexes in vitro. We propose that Pan1 and the type I myosins interactions promote an actin activity important at a late stage in endocytic internalization.

Targeting and processing of membrane-anchored YFP fusion proteins to protein storage vacuoles in transgenic tobacco seeds

2005 ◽  
Vol 15 (4) ◽  
pp. 361-364 ◽  
Author(s):  
Ka Leung Fung ◽  
Yin Fun Yim ◽  
Yu Chung Tse ◽  
Yansong Miao ◽  
Samuel S.M. Sun ◽  
...  
Keyword(s):  
Transgenic Tobacco ◽  
Fusion Proteins ◽  
Seed Protein ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Protein Storage ◽  
Protein Storage Vacuoles ◽  
Pharmaceutical Proteins ◽  
Tobacco Seeds ◽  
Foreign Proteins

Seeds that store proteins in protein storage vacuoles are attractive bioreactors for producing and storing large amounts of pharmaceutical proteins. However, foreign proteins expressed in transgenic plants are subjected to the delivery and modification processes present within plant cells. Here, it is demonstrated that unique membrane sequences deliver a yellow fluorescent protein (YFP) to the seed protein storage vacuoles in transgenic tobacco (Nicotiana tabacum L.) plants, where the YFP is then separated from its membrane anchors. This precise targeting and separation is required for the successful delivery of useful proteins to seed protein storage vacuoles for their stable accumulation in transgenic crops.

Mutational Analysis of the CBFβ-SMMHC Assembly Competence Domain Identifies a Surface Critical for Multimerization and Inhibition of RUNX1/AML1.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2853-2853
Author(s):  
Linsheng Zhang ◽  
Jenice D’Costa ◽  
Tanawan Kummalue ◽  
Isabel Moreno ◽  
Curt I. Civin ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Mutational Analysis ◽  
Coiled Coil ◽  
Hydrophobic Surface ◽  
Homologous Region ◽  
Salt Bridges ◽  
C Terminus ◽  
Myeloid Progenitors

Abstract CBFβ complexes with RUNX1/AML1 to form Core Binding Factor. CBFβ-SMMHC is expressed from the inv(16) or t(16;16) chromosome in 8% of AML cases. This fusion protein contains the majority of CBFβ linked to the α-helical rod domain of smooth muscle myosin heavy chain. CBFβ-SMMHC is thought to contribute to leukemogensis by dominantly inhibiting RUNX1/AML1. Inhibition of AML1 depends upon the integrity of a 28 amino acid region near the C-terminus of the SMMHC segment termed the Assembly Competence Domain (ACD). A homologous region is present in multiple myosins and is required for optimal multimerization of their respective rod domains. The ACD is located within a 63 residue "extended" ACD, which includes 12 residues N-terminal and 23 residues C-terminal to the ACD. The extended ACD was noted to have a more neutral charge than other segments of myosin rods. We have now carried out a mutagenic analysis of individual α-helices within or near the extended ACD and have assessed the effect of these mutations on the ability of CBFβ-SMMHC to multimerize in vitro and to inhibit endogenous AML1 activities in the Ba/F3 cell line and in normal murine myeloid progenitors. The 7 amino acids constituting a single turn of the rod domain α-helix are designated abcdefg. The a and d residues form a hydrophobic surface that mediates coiled-coil dimerization, the e and g residues often form salt bridges that stabilize the dimer, and the b, c and f residues are on the outer surface of the helix and are the best candidates for mediating multimerization. We have therefore mutated the bcf residues as a group in ten helices, N3, N1, A, B, C, D, E, F, G, and H. A–D constitutes the core, 28 residue ACD. N3 and N1 are three or one helix N-terminal to helix A. Mutation of N3 or N1 did not affect multimerization in low ionic strength or the ability of CBFβ-SMMHC to inhibit AML1-mediated G1 to S cell cycle progression in Ba/F3 cells. In contrast, mutation of helices A, B, C, D, E, F, G, or H both impaired multimerization in vitro and prevented cell cycle slowing in Ba/F3 cells. Mutants A–E are each located predominantly in the cell nucleus. In transduced murine myeloid progenitors, mutant N3 again behaved similar to intact CBFβ-SMMHC, mutant A also markedly slowed proliferation, mutant B had an intermediate effect, and mutants C, D, or E did not slow proliferation, each in three independent experiments. The increased activities of mutants A or B in the latter setting may reflect the fact that Ba/F3 cells accumulate three times faster than myeloid progenitors and so perhaps are more sensitive to subtle effects. Sin3A, a co-repressor shown to interact with CBFβ-SMMHC, retained the ability to bind mutants A–E. Analysis of mutants N1 and F–H for mSin3A binding, nuclear localization, and their effects on normal progenitor proliferation is in progress. Together, these findings indicate that a surface near the C-terminus of the CBFβ-SMMHC rod domain, encompassing much of the "extended ACD", is required for multimerization and inhibition of AML1. Helices N1 and H demarcate the boundaries of this surface, with helix H been the very last helix of the rod domain. Further characterization of the molecular interactions which allow this surface to mediate SMMHC multimerization may enable the rationale design of drugs for the therapy of AML associated with inv(16).

scholarly journals Conventional Kinesin Mediates Microtubule-Microtubule Interactions In Vivo

2006 ◽  
Vol 17 (2) ◽  
pp. 907-916 ◽  
Author(s):  
Anne Straube ◽  
Gerd Hause ◽  
Gero Fink ◽  
Gero Steinberg
Keyword(s):  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Binding Activity ◽  
C Terminus ◽  
Cross Bridges ◽  
Conventional Kinesin ◽  
Motor Head

Conventional kinesin is a ubiquitous organelle transporter that moves cargo toward the plus-ends of microtubules. In addition, several in vitro studies indicated a role of conventional kinesin in cross-bridging and sliding microtubules, but in vivo evidence for such a role is missing. In this study, we show that conventional kinesin mediates microtubule-microtubule interactions in the model fungus Ustilago maydis. Live cell imaging and ultrastructural analysis of various mutants in Kin1 revealed that this kinesin-1 motor is required for efficient microtubule bundling and participates in microtubule bending in vivo. High levels of Kin1 led to increased microtubule bending, whereas a rigor-mutation in the motor head suppressed all microtubule motility and promoted strong microtubule bundling, indicating that kinesin can form cross-bridges between microtubules in living cells. This effect required a conserved region in the C terminus of Kin1, which was shown to bind microtubules in vitro. In addition, a fusion protein of yellow fluorescent protein and the Kin1tail localized to microtubule bundles, further supporting the idea that a conserved microtubule binding activity in the tail of conventional kinesins mediates microtubule-microtubule interactions in vivo.

scholarly journals C-terminal tripeptide Ser-Asn-Leu (SNL) of human D-aspartate oxidase is a functional peroxisome-targeting signal

1998 ◽  
Vol 336 (2) ◽  
pp. 367-371 ◽  
Author(s):  
Leen AMERY ◽  
Chantal BREES ◽  
Myriam BAES ◽  
Chiaki SETOYAMA ◽  
Retsu MIURA ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Amino Acid ◽  
Fluorescent Protein ◽  
Consensus Sequence ◽  
C Terminus ◽  
Targeting Signal ◽  
Fluorescent Pattern ◽  
Green Fluorescent ◽  
Positively Charged

The functionality of the C-terminus (Ser-Asn-Leu; SNL) of human d-aspartate oxidase, an enzyme proposed to have a role in the inactivation of synaptically released d-aspartate, as a peroxisome-targeting signal (PTS1) was investigated in vivoand in vitro. Bacterially expressed human d-aspartate oxidase was shown to interact with the human PTS1-binding protein, peroxin protein 5 (PEX5p). Binding was gradually abolished by carboxypeptidase treatment of the oxidase and competitively inhibited by a Ser-Lys-Leu (SKL)-containing peptide. After transfection of mouse fibroblasts with a plasmid encoding green fluorescent protein (GFP) extended by PKSNL (the C-terminal pentapeptide of the oxidase), a punctate fluorescent pattern was evident. The modified GFP co-localized with peroxisomal thiolase as shown by indirect immunofluorescence. On transfection in fibroblasts lacking PEX5p receptor, GFP–PKSNL staining was cytosolic. Peroxisomal import of GFP extended by PGSNL (replacement of the positively charged fourth-last amino acid by glycine) seemed to be slower than that of GFP–PKSNL, whereas extension by PKSNG abolished the import of the modified GFP. Taken together, these results indicate that SNL, a tripeptide not fitting the PTS1 consensus currently defined in mammalian systems, acts as a functional PTS1 in mammalian systems, and that the consensus sequence, based on this work and that of other groups, has to be broadened to (S/A/C/K/N)-(K/R/H/Q/N/S)-L.

Comparative biochemical and morphological changes in imbibed cotton seed hypocotyls and radicles in situ and in vitro – Protein breakdown and elongation growth

1995 ◽  
Vol 5 (1) ◽  
pp. 41-51 ◽  
Author(s):  
Eugene L. Vigil ◽  
Tung K. Fang
Keyword(s):  
Matrix Protein ◽  
Storage Proteins ◽  
Morphological Changes ◽  
Fold Increase ◽  
Hypocotyl Length ◽  
Cortical Cells ◽  
Protein Storage Vacuoles ◽  
Sds Page ◽  
Cotton Seeds

AbstractAxes, hypocotyls and radicles excised from dry cotton seeds (Gossypium hirsutum L. cv. M-8, a double haploid) were imbibed for 24 h and compared with axial segments (excised sections of embryos below the cotyledons) of imbibed, intact seeds. Radicles of excised axes had a 7.4-fold increase in length compared with only 5.2- and 5.7-fold increases, respectively, in radicles of intact seeds and in those isolated when dry. Change in hypocotyl length was not as extensive. EM data for hypocotyl and radicle cortical cells from dry and imbibed seeds revealed a major reduction in matrix protein in protein storage vacuoles along with significant organelle development at 24 h from the start of imbibition. This occurred in parallel with a reduction in salt-extracted proteins and an increase in 2% SDS-extractable proteins. SDS-PAGE of protein from low (0.2 M NaCI) and high (1.0 M NaCI) salt extracts showed a reduction in amount of the major storage proteins (53 and 48 kDa), these bands being almost totally absent in gels of protein extracts from imbibed radicles and significantly reduced in hypocotyls, within 24 h from the start of imbibition. These results indicate that initial elongation of hypocotyls and radicles in intact seeds or of excised axes, after 24 h imbibition, involves breakdown of storage proteins in these axial parts to supply nutrients for growth, with very limited contribution from the cotyledons.

scholarly journals Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’ mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal ‘skip’

2001 ◽  
Vol 82 (5) ◽  
pp. 1013-1025 ◽  
Author(s):  
Michelle L. L. Donnelly ◽  
Garry Luke ◽  
Amit Mehrotra ◽  
Xuejun Li ◽  
Lorraine E. Hughes ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Foot And Mouth Disease ◽  
Peptide Bond ◽  
Open Reading Frame ◽  
Translation Product ◽  
Reading Frame ◽  
C Terminus ◽  
Mouth Disease Virus ◽  
Green Fluorescent

The 2A region of the aphthovirus foot-and-mouth disease virus (FMDV) polyprotein is only 18 aa long. A ‘primary’ intramolecular polyprotein processing event mediated by 2A occurs at its own C terminus. FMDV 2A activity was studied in artificial polyproteins in which sequences encoding reporter proteins flanked the 2A sequence such that a single, long, open reading frame was created. The self-processing properties of these artificial polyproteins were investigated and the co-translational ‘cleavage’ products quantified. The processing products from our artificial polyprotein systems showed a molar excess of ‘cleavage’ product N-terminal of 2A over the product C-terminal of 2A. A series of experiments was performed to characterize our in vitro translation systems. These experiments eliminated the translational or transcriptional properties of the in vitro systems as an explanation for this imbalance. In addition, the processing products derived from a control construct encoding the P1P2 region of the human rhinovirus polyprotein, known to be proteolytically processed, were quantified and found to be equimolar. Translation of a construct encoding green fluorescent protein (GFP), FMDV 2A and β-glucuronidase, also in a single open reading frame, in the presence of puromycin, showed this antibiotic to be preferentially incorporated into the [GFP2A] translation product. We conclude that the discrete translation products from our artificial polyproteins are not produced by proteolysis. We propose that the FMDV 2A sequence, rather than representing a proteolytic element, modifies the activity of the ribosome to promote hydrolysis of the peptidyl(2A)-tRNAGly ester linkage, thereby releasing the polypeptide from the translational complex, in a manner that allows the synthesis of a discrete downstream translation product to proceed. This process produces a ribosomal ‘skip’ from one codon to the next without the formation of a peptide bond.

scholarly journals Identification of a Hydrophobic Domain of HA2 Essential to Morphogenesis of Helicoverpa armigera Nucleopolyhedrovirus

2008 ◽  
Vol 82 (8) ◽  
pp. 4072-4081 ◽  
Author(s):  
Qian Wang ◽  
Yun Wang ◽  
Changyong Liang ◽  
Jianhua Song ◽  
Xinwen Chen
Keyword(s):  
Helicoverpa Armigera ◽  
Fluorescent Protein ◽  
Hydrophobic Domain ◽  
C Terminus ◽  
Localization Analysis ◽  
Helicoverpa Armígera ◽  
Budded Virus ◽  
Green Fluorescent ◽  
Vitro Analysis

ABSTRACT The HA2 protein of the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HearNPV) is a WASP homology protein capable of nucleating branched actin filaments in the presence of the Arp2/3 complex in vitro. To determine the role of ha2 in the HearNPV life cycle, ha2 knockout and ha2 repair bacmids were constructed. Transfection and infection analysis demonstrated that the ha2 null bacmid was unable to produce infectious budded virus (BV), while the repair bacmid rescued the defect. In vitro analysis demonstrated that the WCA domain of HA2 accelerates Arp2/3-mediated actin assembly and is indispensable to the function of HA2. However, analysis of the repaired recombinant with a series of truncated ha2 mutants demonstrated that the WCA domain was essential but not enough to yield infectious virions, and a hydrophobic domain (H domain) consisting of amino acids (aa) 167 to 193 played a pivotal role in the production of BV. Subcellular localization analysis with enhanced green fluorescent protein fusions showed that the H domain functioned as a nuclear localization signal. In addition, deletion of the C terminus of the ha2 product, a phosphatidylinositol 4-kinase homolog, dramatically decreased the viral titer, while deletion of 128 aa from the N terminus did not affect HA2 function.

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