Avital Yahalom

Translational Regulation via 5′ mRNA Leader Sequences Revealed by Mutational Analysis of the Arabidopsis Translation Initiation Factor Subunit eIF3h

Eukaryotic translation initiation factor 3 (eIF3) consists of core subunits that are conserved from yeast to man as well as less conserved, noncore, subunits with potential regulatory roles. Whereas core subunits tend to be indispensable for cell growth, the roles of the noncore subunits remain poorly understood. We addressed the hypothesis that eIF3 noncore subunits have accessory functions that help to regulate translation initiation, by focusing on the Arabidopsis thaliana eIF3h subunit. Indeed, eIF3h was not essential for general protein translation. However, results from transient expression assays and polysome fractionation indicated that the translation efficiency of specific 5′ mRNA leader sequences was compromised in an eif3h mutant, including the mRNA for the basic domain leucine zipper (bZip) transcription factor ATB2/AtbZip11, translation of which is regulated by sucrose. Among other pleiotropic developmental defects, the eif3h mutant required exogenous sugar to transit from seedling to vegetative development, but it was hypersensitive to elevated levels of exogenous sugars. The ATB2 mRNA was rendered sensitive to the eIF3h level by a series of upstream open reading frames. Moreover, eIF3h could physically interact with subunits of the COP9 signalosome, a protein complex implicated primarily in the regulation of protein ubiquitination, supporting a direct biochemical connection between translation initiation and protein turnover. Together, these data implicate eIF3 in mRNA-associated translation initiation events, such as scanning, start codon recognition, or reinitiation and suggest that poor translation initiation of specific mRNAs contributes to the pleiotropic spectrum of phenotypic defects in the eif3h mutant.

Maize mesocotyl plasmodesmata proteins cross-react with connexin gap junction protein antibodies.

Polypeptide present in various cell fractions obtained from homogenized maize mesocotyls were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotted, and screened for cross-reactivity with antibodies against three synthetic polypeptides spanning different regions of the rat heart gap junctional protein connexin43 and the whole mouse liver gap junctional protein connexin32. An antibody raised against a cytoplasmic loop region of connexin43 cross-reacted strongly with a cell wall-associated polypeptide (possibly a doublet) of 26 kilodaltons. Indirect immunogold labeling of thin sections of mesocotyl tissue with this antibody labeled the plasmodesmata of cortical cells along the entire length of the plasmodesmata, including the neck region and the cytoplasmic annulus. Sections labeled with control preimmune serum were essentially free of colloidal gold. An antibody against connexin32 cross-reacted with a 27-kilodalton polypeptide that was present in the cell wall and membrane fractions. Indirect immunogold labeling of thin sections with this antibody labeled the plasmodesmata mainly in the neck region. It is suggested that maize mesocotyl plasmodesmata contain at least two different proteins that have homologous domains with connexin proteins.

The Arabidopsis homologue of an eIF3 complex subunit associates with the COP9 complex

The Arabidopsis COP9 complex is a multi‐subunit repressor of photomorphogenesis which is conserved among multicellular organisms. Approximately 12 proteins copurify with the COP9 complex. Seven of these proteins are orthologues of subunits of the recently published mammalian COP9 complex. Four of the proteins show amino acid similarity to various subunits of the COP9 complex, eIF3 complex and 19S cap of the proteasome. We have studied one of these proteins in order to determine if it is a component of the COP9 complex. Arabidopsis p105 is highly similar to the p110 subunit of the human eIF3. The p105 gene is induced during photomorphogenesis, and RNA and protein analysis reveal different tissue accumulation patterns. p105 is found in a large protein complex. p105 interacts in yeast with both COP9 and FUS6, two known components of the COP9 complex. Our results indicate that p105 is not a component of the COP9 core complex, though it may interact with components of the complex.

A 41 kDa protein isolated from maize mesocotyl cell walls immunolocalizes to plasmodesmata

SummaryPlasmodesmata, dynamic pore structures that traverse plant cell walls, function in cytoplasmic transport between contiguous cells. Cell walls containing embedded plasmodesmata were isolated from mesocotyls of etiolated maize seedlings. Proteins associated with the isolated walls were separated by SDS-PAGE and antibodies were generated against a 41 kDa protein, one of several associated with this wall fraction. Immunoblot analysis showed that the 41 kDa polypeptide was also associated with other subcellular fractions obtained following tissue homogenization and differential centrifugation. The wall associated 41 kDa protein is apparently a peripheral membrane protein since it could be extracted by high salt and high pH. Silver-enhanced immunogold light microscopy showed that the 41 kDa protein was associated with the cell walls of cells both in the stele and cortex. The immunolabeling pattern was transwall and punctate. Electron microscopic immuno-gold labeling localized the polypeptide to plasmodesmata and to electron dense cytoplasmic structures that are apparently Golgi membranes. The significance of the presence of this protein in the Golgi is discussed.

A calcium-dependent protein kinase is associated with maize mesocotyl plasmodesmata

Summary Plasmodesmata (Pd) are trans-wall membrane lined tunnels that regulate cell-to-cell cytoplasmic movement. It has been suggested that Pd conductivity may be regulated by a phosphorylation mechanism. In a maize ( Zea mays L.) mesocotyl cell wall fraction, a Ca 2+ -dependent protein kinase (CDPK) is present that phosphorylates approximately 8 of 20 wall-associated proteins. The kinase is membrane-associated and is not extracted by EGTA, NaCl, up to 4mol/L LiCl, Triton X-100, or Na 2 CO 3 (pH 11), but is fully extracted with SDS or 8 mol/L LiCl. Two polypeptides in the cell wall fraction, with apparent molecular masses of 51 and 56 kD, cross-react with an Arabidopsis CDPK anti-serum and undergo in situ Ca 2+ -dependent autophosphorylation on nitrocellulose. The molecular masses of the CDPKs extracted by 8 mol/L LiCl from the cell wall fraction are different from those extracted from the cell membrane fraction, suggesting that the wall-associated CDPK is unique to the cell wall fraction. Immuno-fluorescence microscopy with isolated walls localizes CDPK to discrete punctate loci in the cell wall. Isolated Pd challenged with CDPK anti-serum show a pattern of cross-reactivity similar to the cell wall fraction. These data suggest that the cell wall-associated CDPK is a putative plasmodesmal-associated membrane protein and may be involved in regulating Pd conductivity.

Isolation and characterization of plasmodesmata.

Publisher Summary This chapter discusses the isolation and characterization of plasmodesmata. Plasmodesmata are dynamic membrane specializations that traverse plant cell walls forming aqueous channels linking adjacent cells. The plasmodesma—like its animal counterpart, the gap junction—functions in the cytoplasmic movement of metabolites and ions. There is strong evidence that plasmodesmata may function to provide a mechanism for intercellular signaling. In addition to their normal physiological function, plasmodesmata can be altered and exploited by viruses as conduits for viral spread from cell to cell. There are also some indications that plasmodesmata in companion cells may be specialized, allowing for the transport of proteins from the companion cells to sieve tube elements. The outer limit of a plasmodesma is formed by the plasmalemma, which is continuous from cell to cell. Within the interior of the plasmalemma tubular envelope runs a strand of modified endoplasmic reticulum that is apparently appressed as seen in static endoplasmic reticulum (ER) micrograph studies and has been termed by various workers as “desmotubule” or “appressed endoplasmic reticulum.”

Arabidopsis eIF3e interacts with subunits of the ribosome, Cop9 signalosome and proteasome

The roles of individual Eukaryotic translation Initiation Factor 3 (eIF3) subunits are largely unclear. Though some are essential, while others are thought to have regulatory roles. The “e” subunit, also known as Int-6, is a candidate for a regulatory subunit as it is not essential for translation initiation in yeasts. To further elucidate the roles of eIF3e, we have employed an interaction-trap screen using the yeast two-hybrid system. eIF3e interacts in yeast with subunits of the ribosome, COP9 signalosome and 26S proteasome. These interactions mesh well with our recent results which showed that eIF3e is degraded in a CSN-dependent, proteasome-dependent fashion, and inhibits translation when present in excess.

Identification of a Light-regulated Protein Kinase Activity from Seedlings of Arabidopsis thaliana¶

Protein kinase transduction pathways are thought to be involved in light signaling in plants, but other than the photoreceptors, no protein kinase activity has been shown to be light‐regulated in vivo. Using an in‐gel protein kinase assay technique with histone H III SS as an exogenous substrate, we identified a light‐regulated protein kinase activity with an apparent molecular weight ca 50 kDa. The kinase activity increased transiently after irradiation of dark‐grown seedlings with continuous far red light (FR) and blue light (B) and decreased after irradiation with red light (R). The maximal activation was achieved after 30 min to 1 h with FR or B. After irradiation times longer than 2 h, the kinase activity decreased to below the sensitivity level of the assay. In Arabidopsis mutants lacking either the photoreceptors phytochrome A, phytochrome B or the blue‐light receptor cryptochrome 1, kinase activity was undetectable, whereas in the photomorphogenic mutants cop1 and det1 the kinase activity was also observed in the absence of light signals, though still stimulated by B and FR. Interestingly, the R inhibition of the kinase activity was lost in the mutant hy5. Pretreatment with cycloheximide blocked the kinase activity.

The etiolated cucumber hypocotyl weight-growth test: A sensitive, easy and ultrafast bioassay for IAA

A sensitive, easy and fast bioassay is described for the detection of IAA. The bioassay consists of measuring the weight increase of hypocotyl sections from etiolated cucumber seedlings incubated in a simple medium containing 2 mM KCl, 0.1 mM CaCl and 10 mg/l chloramphenicol (pH 7). The sensitivity of the test is comparable to that of the Avena first internode test, exhibiting a significant response at an IAA concentration as low as 1 ng/ml. The bioassay requires only 3 h, and is easy to perform. The slope of the log-linear concentration-response curve is moderately steep giving good accuracy. The assay is insensitive to the pH of the media between 4 to 7 and can be performed in the presence or absence of buffer. The growth of the etiolated hypocotyl sections is insensitive to GA and to kinetin except at high concentrations.

A Systems Approach to the COP9 Signalosome

The COP9 signalosome (CSN) was identified close to a decade ago ([Wei et al., 1994][1]). The biochemical purification of the CSN ([Chamovitz et al., 1996][2]) was a major breakthrough, because it showed that Arabidopsis genetics could be approached biochemically, and it subsequently lead to the