The longin domain regulates subcellular targeting of VAMP7 in Arabidopsis thaliana

SNAREs (soluble N-ethyl-maleimide sensitive factor attachment protein receptors) which locate on the specific organelle membrane assure the correct vesicular transport by mediating specific membrane fusions. SNAREs are referred to as R- or Q-SNAREs on the basis of the amino acid sequence similarities and specific conserved residues. All of the Arabidopsis R-SNAREs have a N-terminal domain, called the longin domain (LD). In this study, we investigated the vacuolar targeting mechanism of Arabidopsis R-SNAREs. The vacuolar localized AtVAMP711 was used as the mother protein of GFP-tagged chimeric proteins joined to several domains such as the LD, the SNARE motif (SNM) and the transmembrane domain (TMD) of other organelle-localized R-SNAREs. The results showed that, whereas the TMD is not relevant for the vacuolar targeting, a complete LD is essential for the vacuolar and subcellular targeting.     

拟南芥SRO蛋白家族的结构及功能分析

许多生物及非生物胁迫都会引起植物的氧化胁迫, 参与植物氧化胁迫反应组分的鉴定备受人们的关注。拟南芥SRO家族成员包括AtRCD1、AtSRO1、AtSRO5等, 调节植物对氧化胁迫的反应。AtSROs参与植物正常的生长发育, 同时在植物应对干旱、盐、重金属等胁迫反应中扮演重要角色。AtSROs存在保守的PARP、RST等特殊功能区, 推测其可能具备蛋白的转录、调节、修饰等功能。文章就拟南芥SRO家族成员的基本状况, 在植物生长发育及应对非生物胁迫反应中的作用进行概述, 为进一步研究AtSROs的生物学功能提供理论基础。     

Systematic analysis of protein subcellular localization and interaction using high-throughput transient transformation of Arabidopsis seedlings

The functional genomics approach requires systematic analysis of protein subcellular distribution and interaction networks, preferably by optimizing experimental simplicity and physiological significance. Here, we present an efficient in planta transient transformation system that allows single or multiple expression of constructs containing various fluorescent protein tags in Arabidopsis cotyledons. The optimized protocol is based on vacuum infiltration of agrobacteria directly into young Arabidopsis seedlings. We demonstrate that Arabidopsis epidermal cells show a subcellular distribution of reference markers similar to that in tobacco epidermal cells, and can be used for co-localization or bi-molecular fluorescent complementation studies. We then used this new system to investigate the subcellular distribution of enzymes involved in sphingolipid metabolism. In contrast to transformation systems using tobacco epidermal cells or cultured Arabidopsis cells, our system provides the opportunity to take advantage of the extensive collections of mutant and transgenic lines available in Arabidopsis. The fact that this assay uses conventional binary vectors and a conventional Agrobacterium strain, and is compatible with a large variety of fluorescent tags, makes it a versatile tool for construct screening and characterization before stable transformation. Transient expression in Arabidopsis seedlings is thus a fast and simple method that requires minimum handling and potentially allows medium- to high-throughput analyses of fusion proteins harboring fluorescent tags in a whole-plant cellular context.     

Peroxisomal protein targeting and identification of peroxisomal targeting signals in cholesterol biosynthetic enzymes

At least three different subcellular compartments, including peroxisomes, are involved in cholesterol synthesis. Recently, it has been demonstrated that peroxisomes contain a number of enzymes involved in cholesterol biogenesis that previously were considered to be cytosolic or located in the endoplasmic reticulum. Peroxisomes have been shown to contain acetoacetyl-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, phosphomevalonate decarboxylase, isopentenyl diphosphate isomerase and FPP synthase. Moreover, the activities of these enzymes are also significantly decreased in liver tissue and fibroblast cells obtained from patients with peroxisomal deficiency diseases. In addition, the cholesterol biosynthetic capacity is severely impaired in cultured skin fibroblasts obtained from patients with peroxisomal deficiency diseases. These findings support the proposal that peroxisomes play an essential role in isoprenoid biosynthesis. This paper presents a review of peroxisomal protein targeting and of recent studies demonstrating the localization of cholesterol biosynthetic enzymes in peroxisomes and the identification of peroxisomal targeting signals in these proteins.     

Characterization of Arabidopsis photolyase enzymes and analysis of their role in protection from ultraviolet-B radiation

DNA photolyases are enzymes which mediate the light-dependent repair (photoreactivation) of UV-induced damage products in DNA by direct reversal of base damage rather than via excision repair pathways. Arabidopsis thaliana contains two photolyases specific for photoreactivation of either cyclobutane pyrimidine dimers (CPDs) or pyrimidine (6-4)pyrimidones (6-4PPs), the two major UV-B-induced photoproducts in DNA. Reduced FADH and a reduced pterin were identified as cofactors of the native Arabidopsis CPD photolyase protein. This is the first report of the chromophore composition of any native class II CPD photolyase protein to our knowledge. CPD photolyase protein levels vary between tissues and with leaf age and are highest in flowers and leaves of 3-5-week-old Arabidopsis plants. White light or UV-B irradiation induces CPD photolyase expression in Arabidopsis tissues. This contrasts with the 6-4PP photolyase protein which is constitutively expressed and not regulated by either white or UV-B light. Arabidopsis CPD and 6-4PP photolyase enzymes can remove UV-B-induced photoproducts from DNA in planta even when plants are grown under enhanced levels of UV-B irradiation and at elevated temperatures although the rate of removal of CPDs is slower at high growth temperatures. These studies indicate that Arabidopsis possesses the photorepair capacity to respond effectively to increased UV-B-induced DNA damage under conditions predicted to be representative of increases in UV-B irradiation levels at the Earth's surface and global warming in the twenty-first century.     

Proteins of diverse function and subcellular location are lysine acetylated in Arabidopsis

Acetylation of the ε-amino group of lysine (Lys) is a reversible posttranslational modification recently discovered to be widespread, occurring on proteins outside the nucleus, in most subcellular locations in mammalian cells. Almost nothing is known about this modification in plants beyond the well-studied acetylation of histone proteins in the nucleus. Here, we report that Lys acetylation in plants also occurs on organellar and cytosolic proteins. We identified 91 Lys-acetylated sites on 74 proteins of diverse functional classes. Furthermore, our study suggests that Lys acetylation may be an important posttranslational modification in the chloroplast, since four Calvin cycle enzymes were acetylated. The plastid-encoded large subunit of Rubisco stands out because of the large number of acetylated sites occurring at important Lys residues that are involved in Rubisco tertiary structure formation and catalytic function. Using the human recombinant deacetylase sirtuin 3, it was demonstrated that Lys deacetylation significantly affects Rubisco activity as well as the activities of other central metabolic enzymes, such as the Calvin cycle enzyme phosphoglycerate kinase, the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase, and the tricarboxylic acid cycle enzyme malate dehydrogenase. Our results demonstrate that Lys acetylation also occurs on proteins outside the nucleus in Arabidopsis (Arabidopsis thaliana) and that Lys acetylation could be important in the regulation of key metabolic enzymes.     

Functional analysis of the Hikeshi-like protein and its interaction with HSP70 in Arabidopsis

Heat shock proteins (HSPs) refold damaged proteins and are an essential component of the heat shock response. Previously, the 70 kDa heat shock protein (HSP70) has been reported to translocate into the nucleus in a heat-dependent manner in many organisms. In humans, the heat-induced translocation of HSP70 requires the nuclear carrier protein Hikeshi. In the Arabidopsis genome, only one gene encodes a protein with high homology to Hikeshi, and we named this homolog Hikeshi-like (HKL) protein. In this study, we show that two Arabidopsis HSP70 isoforms accumulate in the nucleus in response to heat shock and that HKL interacts with these HSP70s. Our histochemical analysis revealed that HKL is predominantly expressed in meristematic tissues, suggesting the potential importance of HKL during cell division in Arabidopsis. In addition, we show that HKL regulates HSP70 localization, and HKL overexpression conferred thermotolerance to transgenic Arabidopsis plants. Our results suggest that HKL plays a positive role in the thermotolerance of Arabidopsis plants and cooperatively interacts with HSP70.     

Deg proteases and their role in protein quality control and processing in different subcellular compartments of the plant cell

Degradation of periplasmic proteins (Deg)/high temperature requirement A (HtrA) proteases are ATP-independent serine endopeptidases found in almost every organism. Database searches revealed that 16 Deg paralogues are encoded by the genome of Arabidopsis thaliana, six of which were experimentally shown to be located in chloroplasts, one in peroxisomes, one in mitochondria and one in the nucleus. Two more Deg proteases are predicted to reside in chloroplasts, five in mitochondria (one of them with a dual chloroplastidial/mitochondrial localization) and the subcellular location of one protein is uncertain. This review summarizes the current knowledge on the role of Deg proteases in maintaining protein homeostasis and protein processing in various subcompartments of the plant cell. The chloroplast Deg proteases are the best examined so far, especially with respect to their role in the degradation of photodamaged photosynthetic proteins and in biogenesis of photosystem II (PSII). A combined action of thylakoid lumen and stroma Deg proteases in the primary cleavage of photodamaged D1 protein from PSII reaction centre is discussed on the basis of a recently resolved crystal structure of plant Deg1. The peroxisomal Deg protease is a processing enzyme responsible for the cleavage of N-terminal peroxisomal targeting signals (PTSs). A. thaliana mutants lacking this enzyme show reduced peroxisomal β-oxidation, indicating for the first time the impact of protein processing on peroxisomal functions in plants. Much less data is available for mitochondrial and nuclear Deg proteases. Based on the available expression data we hypothesize a role in general protein quality control and during acquired heat resistance.     

Functional analysis of the Arabidopsis thaliana GCPE protein involved in plastid isoprenoid biosynthesis

Plastid isoprenoids are synthesized via the 2-C-methyl-D-erythritol 4-phosphate pathway. A few years after its discovery, most of the Escherichia coli genes involved in the pathway have been identified, including gcpE. In this work, we have identified an Arabidopsis thaliana protein with homology to the product of this gene. The plant polypeptide, GCPE, contains two structural domains that are absent in the E. coli protein: an N-terminal extension and a central domain of 30 kDa. We demonstrate that the N-terminal region targets the Arabidopsis protein to chloroplasts in vivo, consistent with its role in plastid isoprenoid biosynthesis. Although the presence of the internal extra domain may have an effect on activity, the Arabidopsis mature GCPE was able to complement a gcpE-defective E. coli strain, indicating the plant protein is a true functional homologue of the bacterial gcpE gene product.     

Plant N-glycan processing enzymes employ different targeting mechanisms for their spatial arrangement along the secretory pathway

The processing of N-linked oligosaccharides in the secretory pathway requires the sequential action of a number of glycosidases and glycosyltransferases. We studied the spatial distribution of several type II membrane-bound enzymes from Glycine max, Arabidopsis thaliana, and Nicotiana tabacum. Glucosidase I (GCSI) localized to the endoplasmic reticulum (ER), alpha-1,2 mannosidase I (ManI) and N-acetylglucosaminyltransferase I (GNTI) both targeted to the ER and Golgi, and beta-1,2 xylosyltransferase localized exclusively to Golgi stacks, corresponding to the order of expected function. ManI deletion constructs revealed that the ManI transmembrane domain (TMD) contains all necessary targeting information. Likewise, GNTI truncations showed that this could apply to other type II enzymes. A green fluorescent protein chimera with ManI TMD, lengthened by duplicating its last seven amino acids, localized exclusively to the Golgi and colocalized with a trans-Golgi marker (ST52-mRFP), suggesting roles for protein-lipid interactions in ManI targeting. However, the TMD lengths of other plant glycosylation enzymes indicate that this mechanism cannot apply to all enzymes in the pathway. In fact, removal of the first 11 amino acids of the GCSI cytoplasmic tail resulted in relocalization from the ER to the Golgi, suggesting a targeting mechanism relying on protein-protein interactions. We conclude that the localization of N-glycan processing enzymes corresponds to an assembly line in the early secretory pathway and depends on both TMD length and signals in the cytoplasmic tail.     

Arabidopsis Qa-SNARE SYP2 proteins localized to different subcellular regions function redundantly in vacuolar protein sorting and plant development

SYP2 proteins are a sub-family of Qa-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) that may be responsible for protein trafficking between pre-vacuolar compartments (PVC) and vacuoles. Arabidopsis thaliana SYP22/VAM3/SGR3 and SYP21/PEP12 proteins function independently, but are both reported to be essential for male gametophytic viability. Here, we systematically examined the redundancy of three SYP2 paralogs (i.e. SYP21, 22 and 23) using a Col-0 ecotype harboring a SYP2 paralog (SYP23/PLP) that lacked a transmembrane domain. Surprisingly, no visible phenotypes were observed, even in the double knockout syp21/pep12 syp23/plp. Deficiency of either SYP21/PEP12 or SYP23/PLP in the syp22 background resulted in a defect in vacuolar protein sorting, characterized by abnormal accumulation of protein precursors in seeds. SYP21/PEP12 knockdown enhanced the syp22 phenotype (i.e. semi-dwarfism, poor leaf vein development and abnormal development of myrosin cells), and additional knockout of SYP23/PLP further aggravated the phenotype. A GFP-SYP23/PLP fusion localized to the cytosol, but not to the PVC or vacuolar membrane, where SYP21/PEP12 or SYP22/VAM3, respectively, were localized. Immunoprecipitation analysis showed that SYP23/PLP interacted with the vacuolar Qb- and Qc-SNAREs, VTI11 and SYP5, respectively, suggesting that SYP23/PLP is able to form a SNARE complex anchoring the membrane. Unexpectedly, we found that expression of multiple copies of a genomic fragment of SYP23/PLP suppressed the abnormal syp22-3 phenotype. Thus, SYP2 proteins, including cytosolic SYP23/PLP, appear to function redundantly in vacuolar trafficking and plant development.     

蛋白质酶功能分析和预测方法的进展和前瞻

蛋白质酶是生物体内最重要的生物分子之一。对酶的功能进行系统研究具有重要的科学研究价值和工业应用意义,近年来,以计算机技术为基础的酶功能预测的方法不断发展与完善。基于此背景,本文总结了基于计算方法的酶功能分析与预测的主要方法,包括酶结合位点、分子对接、动力学模拟以及分子设计等内容。同时,本文也对相应的发展趋势进行讨论和展望。     

Vacuolar processing enzymes are essential for proper processing of seed storage proteins in Arabidopsis thaliana

The proprotein precursors of storage proteins are post-translationally processed to produce their respective mature forms within the protein storage vacuoles of maturing seeds. To investigate the processing mechanism in vivo, we isolated Arabidopsis mutants that accumulate detectable amounts of the precursors of the storage proteins, 12 S globulins and 2 S albumins, in their seeds. All six mutants isolated have a defect in the beta VPE gene. VPE (vacuolar processing enzyme) is a cysteine proteinase with substrate specificity toward an asparagine residue. We further generated various mutants lacking different VPE isoforms: alpha VPE, beta VPE, and/or gamma VPE. More than 90% of VPE activity is abolished in the beta vpe-3 seeds, and no VPE activity is detected in the alpha vpe-1/beta vpe-3/gamma vpe-1 seeds. The triple mutant seeds accumulate no properly processed mature storage proteins. Instead, large amounts of storage protein precursors are found in the seeds of this mutant. In contrast to beta vpe-3 seeds, which accumulate both precursors and mature storage proteins, the other single (alpha vpe-1 and gamma vpe-1) and double (alpha vpe-1/gamma vpe-1) mutants accumulate no precursors in their seeds at all. Therefore, the vegetative VPEs, alpha VPE and gamma VPE, are not necessary for precursor processing in the presence of beta VPE, but partly compensates for the deficiency in beta VPE in beta vpe-3 seeds. In the absence of functional VPEs, a proportion of pro2S albumin molecules are alternatively cleaved by aspartic proteinase. This cleavage by aspartic proteinase is promoted by the initial processing of pro2S albumins by VPE. Our overall results suggest that seed-type beta VPE is most essential for the processing of storage proteins, and that the vegetative-type VPEs and aspartic proteinase complement beta VPE activity in this processing.     

Acyl-CoA synthetase and the peroxisomal enzymes of beta-oxidation in human liver. Quantitative analysis of their subcellular localization.

The presence of acyl-CoA synthetase (EC 6.2.1.3) in peroxisomes and the subcellular distribution of beta-oxidation enzymes in human liver were investigated by using a single-step fractionation method of whole liver homogenates in metrizamide continuous density gradients and a novel procedure of computer analysis of results. Peroxisomes were found to contain 16% of the liver palmitoyl-CoA synthetase activity, and 21% and 60% of the enzyme activity was localized in mitochondria and microsomal fractions respectively. Fatty acyl-CoA oxidase was localized exclusively in peroxisomes, confirming previous results. Human liver peroxisomes were found to contribute 13%, 17% and 11% of the liver activities of crotonase, beta-hydroxyacyl-CoA dehydrogenase and thiolase respectively. The absolute activities found in peroxisomes for the enzymes investigated suggest that in human liver fatty acyl-CoA oxidase is the rate-limiting enzyme of the peroxisomal beta-oxidation pathway, when palmitic acid is the substrate.     

Crystallographic analysis of CaaX prenyltransferases complexed with substrates defines rules of protein substrate selectivity

Post-translational modifications are essential for the proper function of many proteins in the cell. The attachment of an isoprenoid lipid (a process termed prenylation) by protein farnesyltransferase (FTase) or geranylgeranyltransferase type I (GGTase-I) is essential for the function of many signal transduction proteins involved in growth, differentiation, and oncogenesis. FTase and GGTase-I (also called the CaaX prenyltransferases) recognize protein substrates with a C-terminal tetrapeptide recognition motif called the Ca1a2X box. These enzymes possess distinct but overlapping protein substrate specificity that is determined primarily by the sequence identity of the Ca1a2X motif. To determine how the identity of the Ca1a2X motif residues and sequence upstream of this motif affect substrate binding, we have solved crystal structures of FTase and GGTase-I complexed with a total of eight cognate and cross-reactive substrate peptides, including those derived from the C termini of the oncoproteins K-Ras4B, H-Ras and TC21. These structures suggest that all peptide substrates adopt a common binding mode in the FTase and GGTase-I active site. Unexpectedly, while the X residue of the Ca1a2X motif binds in the same location for all GGTase-I substrates, the X residue of FTase substrates can bind in one of two different sites. Together, these structures outline a series of rules that govern substrate peptide selectivity; these rules were utilized to classify known protein substrates of CaaX prenyltransferases and to generate a list of hypothetical substrates within the human genome.     

Functional evolutionary divergence of proteolytic enzymes and their inhibitors

The amino acid residues of protease inhibitors that interact directly with proteases are hypervariable, suggesting recent positive evolutionary selection for functional differences between them. A similar hypervariability is apparent in some proteases, which may be co-evolving with the inhibitors.