Formation of stacked ER cisternae by low affinity protein interactions

The endoplasmic reticulum (ER) can transform from a network of branching tubules into stacked membrane arrays (termed organized smooth ER [OSER]) in response to elevated levels of specific resident proteins, such as cytochrome b(5). Here, we have tagged OSER-inducing proteins with green fluorescent protein (GFP) to study OSER biogenesis and dynamics in living cells. Overexpression of these proteins induced formation of karmellae, whorls, and crystalloid OSER structures. Photobleaching experiments revealed that OSER-inducing proteins were highly mobile within OSER structures and could exchange between OSER structures and surrounding reticular ER. This indicated that binding interactions between proteins on apposing stacked membranes of OSER structures were not of high affinity. Addition of GFP, which undergoes low affinity, antiparallel dimerization, to the cytoplasmic domains of non-OSER-inducing resident ER proteins was sufficient to induce OSER structures when overexpressed, but addition of a nondimerizing GFP variant was not. These results point to a molecular mechanism for OSER biogenesis that involves weak homotypic interactions between cytoplasmic domains of proteins. This mechanism may underlie the formation of other stacked membrane structures within cells.     

Superfolder GFP is fluorescent in oxidizing environments when targeted via the Sec translocon

The ability to study proteins in live cells using genetically encoded fluorescent proteins (FPs) has revolutionized cell biology (1-3). Researchers have created numerous FP biosensors and optimized FPs for specific organisms and subcellular environments in a rainbow of colors (4,5). However, expressing FPs in oxidizing environments such as the eukaryotic endoplasmic reticulum (ER) or the bacterial periplasm can impair folding, thereby preventing fluorescence (6,7). A substantial fraction of enhanced green fluorescent protein (EGFP) oligomerizes to form non-fluorescent mixed disulfides in the ER (6) and EGFP does not fluoresce in the periplasm when targeted via the SecYEG translocon (7). To overcome these obstacles, we exploited the highly efficient folding capability of superfolder GFP (sfGFP) (8). Here, we report sfGFP does not form disulfide-linked oligomers in the ER and maltose-binding protein (MBP) signal sequence (peri)-sfGFP (9) is brightly fluorescent in the periplasm of Escherichia coli. Thus, sfGFP represents an important research tool for studying resident proteins of oxidizing environments.     

含Dsred类似生色团的红色荧光蛋白的发展及应用

自从绿色荧光蛋白(GFP)被发现以来,荧光蛋白在生物医学领域已经成为一种重要的荧光成像工具．随着红色荧光蛋白DsRed的出现,各种优化的DsRed突变体和远红荧光蛋白也不断涌现．其中荧光蛋白生色团的形成机制对改建更优的荧光蛋白变种影响很大,对于红色荧光蛋白而言,大多数的红色荧光蛋白的生色团类型为DsRed类似生色团,在此基础上又出现了Far-red DsRed类似生色团．目前,含DsRed类似生色团的荧光蛋白主要有单体红色荧光蛋白、光转换荧光蛋白、斯托克斯红移蛋白、荧光计时器等．这些优化的荧光蛋白作为分子探针可以实现对活细胞、细胞器或胞内分子的时空标记和追踪,已经在生物工程学、细胞生物学、基础医学领域得到广泛应用．本文综述了含DsRed类似生色团的荧光蛋白的研究进展及其应用,以及由此发展起来的远红荧光蛋白在活体显微成像技术中的应用,并展望了荧光探针技术研究的新方向．     

A Monomeric Photoconvertible Fluorescent Protein for Imaging of Dynamic Protein Localization

The use of green-to-red photoconvertible fluorescent proteins (FPs) enables researchers to highlight a subcellular population of a fusion protein of interest and to image its dynamics in live cells. In an effort to enrich the arsenal of photoconvertible FPs and to overcome the limitations imposed by the oligomeric structure of natural photoconvertible FPs, we designed and optimized a new monomeric photoconvertible FP. Using monomeric versions of Clavularia sp. cyan FP as template, we employed sequence-alignment-guided design to create a chromophore environment analogous to that shared by known photoconvertible FPs. The designed gene was synthesized and, when expressed in Escherichia coli, found to produce green fluorescent colonies that gradually switched to red after exposure to white light. We subjected this first-generation FP [named mClavGR1 (monomeric Clavularia-derived green-to-red photoconvertible 1)] to a combination of random and targeted mutageneses and screened libraries for efficient photoconversion using a custom-built system for illuminating a 10-cm Petri plate with 405-nm light. Following more than 15 rounds of library creation and screening, we settled on an optimized version, known as mClavGR2, that has eight mutations relative to mClavGR1. Key improvements of mClavGR2 relative to mClavGR1 include a 1.4-fold brighter red species, 1.8-fold higher photoconversion contrast, and dramatically improved chromophore maturation in E. coli. The monomeric status of mClavGR2 has been demonstrated by gel-filtration chromatography and the functional expression of a variety of mClavGR2 chimeras in mammalian cells. Furthermore, we have exploited mClavGR2 to determine the diffusion kinetics of the membrane protein intercellular adhesion molecule 1 both when the membrane is in contact with a T-lymphocyte expressing leukocyte-function-associated antigen 1 and when it is not. These experiments clearly establish that mClavGR2 is well suited for rapid photoconversion of protein subpopulations and subsequent tracking of dynamic changes in localization in living cells.     

IntEResting structures: formation and applications of organized smooth endoplasmic reticulum in plant cells

The endoplasmic reticulum (ER) is an organelle with remarkable plasticity, capable of rapidly changing its structure to accommodate different functions based on intra- and extracellular cues. One of the ER structures observed in plants is known as “organized smooth endoplasmic reticulum” (OSER), consisting of symmetrically stacked ER membrane arrays. In plants, these structures were first described in certain specialized tissues, e.g. the sieve elements of the phloem, and more recently in transgenic plants overexpressing ER membrane resident proteins. To date, much of the investigation of OSER focused on yeast and animal cells but research into plant OSER has started to grow. In this update, we give a succinct overview of research into the OSER phenomenon in plant cells with case studies highlighting both native and synthetic occurrences of OSER. We also assess the primary driving forces that trigger the formation of OSER, collating evidence from the literature to compare two competing theories for the origin of OSER: that OSER formation is initiated by oligomerizing protein accumulation in the ER membrane or that OSER is the result of ER membrane proliferation. This has long been a source of controversy in the field and here we suggest a way to integrate arguments from both sides into a single unifying theory. Finally, we discuss the potential biotechnological uses of OSER as a tool for the nascent plant synthetic biology field with possible applications as a synthetic microdomain for metabolic engineering and as an extensive membrane surface for synthetic chemistry or protein accumulation.

Novel findings in organized smooth endoplasmic reticulum uncover mechanisms of formation and potential applications for this structure.     

Sertoli cell behaviors in developing testis cords and postnatal seminiferous tubules of the mouse

Sertoli cells are the primary structural component of the fetal testis cords and postnatal seminiferous tubules. Live imaging technologies facilitate the visualization of cell morphologies and behaviors through developmental processes. A transgenic mouse line was generated using a fragment of the rat Gata4 gene to direct the expression of a dual-color fluorescent protein reporter in fetal and adult Sertoli cells. The reporter encoded a red fluorescent protein, monomeric Cherry (mCherry), fused to histone 2B and enhanced green fluorescent protein (EGFP) fused to a glycosylphosphatidylinositol sequence, with a self-cleaving 2A polypeptide separating the two fusion proteins. After translation, the red and green fluorescent proteins translocated to the nucleus and plasma membrane, respectively, of Sertoli cells. Transgene expression in testes was first detected by fluorescent microscopy around Embryonic Day 12.0. Sertoli cell division and migration were visualized during testis cord formation in organ culture. Initially, the Sertoli cells had mesenchyme-like morphologies and behaviors, but later, the cells migrated to the periphery of the testis cords to become epithelialized. In postnatal seminiferous tubules, Sertoli nuclei were evenly spaced when viewed from the external surface of tubules, and Sertoli cytoplasm and membranes were associated with germ cells basally in a rosette pattern. This mouse line was bred to previously described transgenic mouse lines expressing EGFP in Sertoli cytoplasm or a nuclear cyan fluorescent protein (Cerulean) and mCherry in plasma membranes of germ cells. This revealed the physical relationship between Sertoli and germ cells in developing testis cords and provided a novel perspective on Sertoli cell development.     

The critical role of disulfide bond formation in protein sorting in the endosperm of rice

Many seed storage proteins, including monomeric 2S albumin and polymeric prolamin, contain conserved sequences in three separate regions, termed A, B, and C, which contain the consensus motifs LxxC, CCxQL, and PxxC, respectively. Protein-sorting mechanisms in rice (Oryza sativa) endosperm were studied with a green fluorescent protein (GFP) fused to different segments of rice α-globulin, a monomeric, ABC-containing storage protein. The whole ABC region together with GFP was efficiently transported to protein storage vacuoles (type II protein bodies [PB-II]) in the endosperm cells and sequestered in the matrix that surrounds the crystalloids. Peptide Gln-23 to Ser-43 in the A region was sufficient to guide GFP to PB-II. However, GFP fused with the AB or B region accumulated in prolamin protein bodies. Substitution mutations in the CCxQL motif in the B region significantly altered protein localization in the endosperm cells. Furthermore, protein extracts containing these substituted proteins had increased amounts of the endoplasmic reticulum (ER) chaperons BiP (for binding protein), protein disulfide isomerase, and calnexin as a part of protein complexes that were insoluble in a detergent buffer. These results suggest that the ER chaperons and disulfide bonds formed at the dicysteine residues in CCxQL play critical roles in sorting fused proteins in the endosperm cells.     

mEosFP-based green-to-red photoconvertible subcellular probes for plants

Photoconvertible fluorescent proteins (FPs) are recent additions to the biologists' toolbox for understanding the living cell. Like green fluorescent protein (GFP), monomeric EosFP is bright green in color but is efficiently photoconverted into a red fluorescent form using a mild violet-blue excitation. Here, we report mEosFP-based probes that localize to the cytosol, plasma membrane invaginations, endosomes, prevacuolar vesicles, vacuoles, the endoplasmic reticulum, Golgi bodies, mitochondria, peroxisomes, and the two major cytoskeletal elements, filamentous actin and cortical microtubules. The mEosFP fusion proteins are smaller than GFP/red fluorescent protein-based probes and, as demonstrated here, provide several significant advantages for imaging of living plant cells. These include an ability to differentially color label a single cell or a group of cells in a developing organ, selectively highlight a region of a cell or a subpopulation of organelles and vesicles within a cell for tracking them, and understanding spatiotemporal aspects of interactions between similar as well as different organelles. In addition, mEosFP probes introduce a milder alternative to fluorescence recovery after photobleaching, whereby instead of photobleaching, photoconversion followed by recovery of green fluorescence can be used for estimating subcellular dynamics. Most importantly, the two fluorescent forms of mEosFP furnish bright internal controls during imaging experiments and are fully compatible with cyan fluorescent protein, GFP, yellow fluorescent protein, and red fluorescent protein fluorochromes for use in simultaneous, multicolor labeling schemes. Photoconvertible mEosFP-based subcellular probes promise to usher in a much higher degree of precision to live imaging of plant cells than has been possible so far using single-colored FPs.     

NHE6 protein possesses a signal peptide destined for endoplasmic reticulum membrane and localizes in secretory organelles of the cell

The NHE6 protein is a unique Na(+)/H(+) exchanger isoform believed to localize in mitochondria. It possesses a hydrophilic N-terminal portion that is rich in positively charged residues and many hydrophobic segments. In the present study, signal sequences in the NHE6 molecule were examined for organelle localization and membrane topogenesis. When the full-length protein was expressed in COS7 cells, it localized in the endoplasmic reticulum and on the cell surface. Furthermore, the protein was fully N-glycosylated. When green fluorescent protein was fused after the second (H2) or third (H3) hydrophobic segment, the fusion proteins were targeted to the endoplasmic reticulum (ER) membrane. The localization pattern was the same as that of fusion proteins in which green fluorescent protein was fused after H2 of NHE1. In an in vitro system, H1 behaved as a signal peptide that directs the translocation of the following polypeptide chain and is then processed off. The next hydrophobic segment (H2) halted translocation and eventually became a transmembrane segment. The N-terminal hydrophobic segment (H1) of NHE1 also behaved as a signal peptide. Cell fractionation studies using antibodies against the 15 C-terminal residues indicated that NHE6 protein localized in the microsomal membranes of rat liver cells. All of the NHE6 molecules in liver tissue possess an endoglycosidase H-resistant sugar chain. These findings indicate that NHE6 protein is targeted to the ER membrane via the N-terminal signal peptide and is sorted to organelle membranes derived from the ER membrane.     

A redox-sensitive luciferase assay for determining the localization and topology of endoplasmic reticulum proteins

Correct localization and transmembrane topology are crucial for the proteins residing and functioning in the endoplasmic reticulum (ER). We have developed a rapid and convenient assay, based on the redox-sensitive luciferase from Gaussia princeps (Gluc) and green fluorescence protein (GFP), to determine the localization or topology of ER proteins. Using the tandem Gluc-GFP reporter fused to different positions of a target protein, we successfully characterized the topologies of two ER transmembrane proteins Herp and HRD1 that are involved in the ER quality control system. This assay method may also be applicable to the proteins in secretory pathway, plasma membrane, and other compartments of cells.     

A green-emitting fluorescent protein from Galaxeidae coral and its monomeric version for use in fluorescent labeling

We have cloned a gene which encodes a fluorescent protein from the stony coral, Galaxeidae. This protein absorbs light maximally at 492 nm and emits green light at 505 nm, and as a result, we have designated it "Azami-Green (AG)." Despite sharing a similar spectral profile with enhanced green fluorescent protein (EGFP) (Clontech), the most popular variant of the Aequorea victoria green fluorescent protein, the identity between these two proteins at the amino acid level is only 5.7%. However, since AG has a high extinction coefficient, fluorescence quantum yield, and acid stability, it produces brighter green fluorescence in cultured cells than EGFP. Similar to other fluorescent proteins isolated from coral animals, AG forms a tight tetrameric complex, resulting in poor labeling of subcellular structures such as the plasma membrane and mitochondria. We have converted tetrameric AG into a monomeric form by the introduction of three amino acid substitutions, which were recently reported to be effective for monomerizing the red fluorescent protein from Discosoma coral (DsRed, Clontech). The resultant monomeric AG allowed for efficient fluorescent labeling of all of the subcellular structures and proteins tested while retaining nearly all of the brightness of the original tetrameric form. Thus, monomeric AG is a useful monomeric green-emitting fluorescent protein comparable to EGFP.     

Expression of the sarco/endoplasmic Ca(2+)-ATPase, SERCA1a, in fibroblasts induces the formation of organelle membrane arrays

Members of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) family are transmembrane proteins that are essential for the function of intracellular Ca(2+) storage organelles. We found that overexpression of avian muscle SERCA1a in transfected mouse fibroblasts led to the appearance of tubular membrane bundles that we termed plaques. These structures were generated in transfected cells when SERCA1a protein expression approached the endogenous level measured in chicken skeletal muscle. Plaque membranes had associated ribosomes and contained endoplasmic reticulum (ER) proteins. Endogenous ER protein levels were not elevated in SERCA1a-expressing cells, indicating that plaques were not generalized proliferations of ER but rather a reorganization of existing organelle membrane. Plaque formation also was observed in cells expressing a green fluorescent protein-SERCA1a fusion protein (GFP-SERCA1a). GFP-SERCA1a molecules displayed extensive lateral mobility between plaques, suggesting the presence of membrane continuities between these structures. Plaques were induced in cells expressing cDNA encoding a catalytically silent SERCA1a mutant indicating that ER redistribution was driven by a structural feature of the enzyme. SERCA1a-induced plaque formation shares some characteristics of sarcoplasmic reticulum (SR) biogenesis during muscle differentiation, and high-level SERCA1a expression in vivo may contribute to the formation of SR from ER during embryonic myogenesis.     

An approach for reducing unwanted oligomerisation of DsRed fusion proteins

Oligomerisation of the red fluorescent protein, DsRed, can interfere with the localisation and function of proteins to which it is fused. We demonstrate an approach that may help to reduce significantly the impact of oligomerisation on the biology of the protein fusion partner. Growth of yeast (Saccharomyces cerevisiae) cells expressing ATP synthase containing subunit gamma-DsRed fusion was compromised relative to control cells. Furthermore, ATP synthase was found to exist as oligomeric structures when isolated under conditions where monomers would normally be present. The compromised growth phenotype was partially reversed and the oligomerisation of the ATP synthase reduced when a non-fluorescent variant of DsRed not fused to another protein was targeted to the mitochondrion in addition to the gamma-DsRed fusion protein. This strategy may also be applicable to the reduction of unwanted interactions between fusion proteins that contain the normally dimeric fluorescent proteins HcRed or Renilla GFP.     

A high-throughput screening of genes that encode proteins transported into the endoplasmic reticulum in mammalian cells

The compartments of eukaryotic cells maintain a distinct protein composition to perform a variety of specialized functions. We developed a new method for identifying the proteins that are transported to the endoplasmic reticulum (ER) in living mammalian cells. The principle is based on the reconstitution of two split fragments of enhanced green fluorescent protein (EGFP) by protein splicing with DnaE from Synechocystis PCC6803. Complementary DNA (cDNA) libraries fused to the N-terminal halves of DnaE and EGFP are introduced in mammalian cells with retroviruses. If an expressed protein is transported into the ER, the N-terminal half of EGFP meets its C-terminal half in the ER, and full-length EGFP is reconstituted by protein splicing. The fluorescent cells are isolated using fluorescence-activated cell sorting and the cDNAs are sequenced. The developed method was able to accurately identify cDNAs that encode proteins transported to the ER. We identified 27 novel proteins as the ER-targeting proteins. The present method overcomes the limitation of the previous GFP- or epitope-tagged methods, using which it was difficult to identify the ER-targeting proteins in a high-throughput manner.     

Dynamic trafficking of wheat γ-gliadin and of its structural domains in tobacco cells, studied with fluorescent protein fusions

Prolamins, the main storage proteins of wheat seeds, are synthesized and retained in the endoplasmic reticulum (ER) of the endosperm cells, where they accumulate in protein bodies (PBs) and are then exported to the storage vacuole. The mechanisms leading to these events are unresolved. To investigate this unconventional trafficking pathway, wheat γ-gliadin and its isolated repeated N-terminal and cysteine-rich C-terminal domains were fused to fluorescent proteins and expressed in tobacco leaf epidermal cells. The results indicated that γ-gliadin and both isolated domains were able to be retained and accumulated as protein body-like structures (PBLS) in the ER, suggesting that tandem repeats are not the only sequence involved in γ-gliadin ER retention and PBLS formation. The high actin-dependent mobility of γ-gliadin PBLS is also reported, and it is demonstrated that most of them do not co-localize with Golgi body or pre-vacuolar compartment markers. Both γ-gliadin domains are found in the same PBLS when co-expressed, which is most probably due to their ability to interact with each other, as indicated by the yeast two-hybrid and FRET-FLIM experiments. Moreover, when stably expressed in BY-2 cells, green fluorescent protein (GFP) fusions to γ-gliadin and its isolated domains were retained in the ER for several days before being exported to the vacuole in a Golgi-dependent manner, and degraded, leading to the release of the GFP 'core'. Taken together, the results show that tobacco cells are a convenient model to study the atypical wheat prolamin trafficking with fluorescent protein fusions.     

Diacidic motif is required for efficient transport of the K+ channel KAT1 to the plasma membrane

For a number of mammalian ion channels, trafficking to the plasma membrane was found to be controlled by intrinsic sequence motifs. Among these sequences are diacidic motifs that function as endoplasmic reticulum (ER) export signals. So far it is unclear if similar motifs also exist in plant ion channels. In this study we analyzed the function of four diacidic DXE/DXD motifs of the plant K(+) channel KAT1. Mutation of the first diacidic DXE motif resulted in a strong reduction of the KAT1 conductance in both guard cell protoplasts and HEK293 cells (human embryonic kidney cells). Confocal fluorescence microscopy of guard cells expressing the mutated KAT1 fused to green fluorescent protein revealed localization of the mutated channel only in intracellular structures around the nucleus. These structures could be identified as part of the ER via coexpression of KAT1 fused to yellow fluorescent protein with an ER-retained protein (HDEL) fused to cyan fluorescent protein. Block of vesicle formation from the ER by overexpression of the small GTP-binding protein Sar1 fixed in its GDP-bound form led to retention of wild-type KAT1 in similar parts of the ER. Mutation of the three other diacidic motifs had no effect. Together, the results demonstrate that one diacidic motif of KAT1 is essential for ER export of the functional channel in both guard cell protoplasts and HEK293 cells. This suggests that trafficking of plant plasma membrane ion channels is controlled via a conserved mechanism.     

Dynamics and retention of misfolded proteins in native ER membranes

When co-translationally inserted into endoplasmic reticulum (ER) membranes, newly synthesized proteins encounter the lumenal environment of the ER, which contains chaperone proteins that facilitate the folding reactions necessary for protein oligomerization, maturation and export from the ER. Here we show, using a temperature-sensitive variant of vesicular stomatitis virus G protein tagged with green fluorescent protein (VSVG-GFP), and fluorescence recovery after photobleaching (FRAP), the dynamics of association of folded and misfolded VSVG complexes with ER chaperones. We also investigate the potential mechanisms underlying protein retention in the ER. Misfolded VSVG-GFP complexes at 40 degrees C are highly mobile in ER membranes and do not reside in post-ER compartments, indicating that they are not retained in the ER by immobilization or retrieval mechanisms. These complexes are immobilized in ATP-depleted or tunicamycin-treated cells, in which VSVG-chaperone interactions are no longer dynamic. These results provide insight into the mechanisms of protein retention in the ER and the dynamics of protein-folding complexes in native ER membranes.     

Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins

Fluorescence anisotropy decay microscopy was used to determine, in individual living cells, the spatial monomer-dimer distribution of proteins, as exemplified by herpes simplex virus thymidine kinase (TK) fused to green fluorescent protein (GFP). Accordingly, the fluorescence anisotropy dynamics of two fusion proteins (TK27GFP and TK366GFP) was recorded in the confocal mode by ultra-sensitive time-correlated single-photon counting. This provided a measurement of the rotational time of these proteins, which, by comparing with GFP, allowed the determination of their oligomeric state in both the cytoplasm and the nucleus. It also revealed energy homo-transfer within aggregates that TK366GFP progressively formed. Using a symmetric dimer model, structural parameters were estimated; the mutual orientation of the transition dipoles of the two GFP chromophores, calculated from the residual anisotropy, was 44.6 +/- 1.6 degrees, and the upper intermolecular limit between the two fluorescent tags, calculated from the energy transfer rate, was 70 A. Acquisition of the fluorescence steady-state intensity, lifetime, and anisotropy decay in the same cells, at different times after transfection, indicated that TK366GFP was initially in a monomeric state and then formed dimers that grew into aggregates. Picosecond time-resolved fluorescence anisotropy microscopy opens a promising avenue for obtaining structural information on proteins in individual living cells, even when expression levels are very low.     

Fluorescent protein biosensors applied to microphysiological systems

This mini-review discusses the evolution of fluorescence as a tool to study living cells and tissues in vitro and the present role of fluorescent protein biosensors (FPBs) in microphysiological systems (MPSs). FPBs allow the measurement of temporal and spatial dynamics of targeted cellular events involved in normal and perturbed cellular assay systems and MPSs in real time. FPBs evolved from fluorescent analog cytochemistry (FAC) that permitted the measurement of the dynamics of purified proteins covalently labeled with environmentally insensitive fluorescent dyes and then incorporated into living cells, as well as a large list of diffusible fluorescent probes engineered to measure environmental changes in living cells. In parallel, a wide range of fluorescence microscopy methods were developed to measure the chemical and molecular activities of the labeled cells, including ratio imaging, fluorescence lifetime, total internal reflection, 3D imaging, including super-resolution, as well as high-content screening. FPBs evolved from FAC by combining environmentally sensitive fluorescent dyes with proteins in order to monitor specific physiological events such as post-translational modifications, production of metabolites, changes in various ion concentrations, and the dynamic interaction of proteins with defined macromolecules in time and space within cells. Original FPBs involved the engineering of fluorescent dyes to sense specific activities when covalently attached to particular domains of the targeted protein. The subsequent development of fluorescent proteins (FPs), such as the green fluorescent protein, dramatically accelerated the adoption of studying living cells, since the genetic “labeling” of proteins became a relatively simple method that permitted the analysis of temporal–spatial dynamics of a wide range of proteins. Investigators subsequently engineered the fluorescence properties of the FPs for environmental sensitivity that, when combined with targeted proteins/peptides, created a new generation of FPBs. Examples of FPBs that are useful in MPS are presented, including the design, testing, and application in a liver MPS.     

Two plant-viral movement proteins traffic in the endocytic recycling pathway

Many plant viruses exploit a conserved group of proteins known as the triple gene block (TGB) for cell-to-cell movement. Here, we investigated the interaction of two TGB proteins (TGB2 and TGB3) of Potato mop-top virus (PMTV), with components of the secretory and endocytic pathways when expressed as N-terminal fusions to green fluorescent protein or monomeric red fluorescent protein (mRFP). Our studies revealed that fluorophore-labeled TGB2 and TGB3 showed an early association with the endoplasmic reticulum (ER) and colocalized in motile granules that used the ER-actin network for intracellular movement. Both proteins increased the size exclusion limit of plasmodesmata, and TGB3 accumulated at plasmodesmata in the absence of TGB2. TGB3 contains a putative Tyr-based sorting motif, mutations in which abolished ER localization and plasmodesmatal targeting. Later in the expression cycle, both fusion proteins were incorporated into vesicular structures. TGB2 associated with these structures on its own, but TGB3 could not be incorporated into the vesicles in the absence of TGB2. Moreover, in addition to localization to the ER and motile granules, mRFP-TGB3 was incorporated into vesicles when expressed in PMTV-infected epidermal cells, indicating recruitment by virus-expressed TGB2. The TGB fusion protein-containing vesicles were labeled with FM4-64, a marker for plasma membrane internalization and components of the endocytic pathway. TGB2 also colocalized in vesicles with Ara7, a Rab5 ortholog that marks the early endosome. Protein interaction analysis revealed that recombinant TGB2 interacted with a tobacco protein belonging to the highly conserved RME-8 family of J-domain chaperones, shown to be essential for endocytic trafficking in Caenorhabditis elegans and Drosophila melanogaster. Collectively, the data indicate the involvement of the endocytic pathway in viral intracellular movement, the implications of which are discussed.