Cdc48 and ubiquilins confer selective anterograde protein sorting and entry into the multivesicular body in yeast.

Cdc48/p97 and the ubiquilin family of UBA-UBL proteins are known for their role in the retrotranslocation of damaged proteins from the endoplasmic reticulum. We demonstrate that Cdc48 and the ubiquilin-like proteins in yeast also play a role in the anterograde trafficking of proteins, in this case the vacuolar protease, Cps1.

Overexpression of a conserved HSP40 chaperone reduces toxicity of several neurodegenerative disease proteins.

TDP-43 and FUS are DNA/RNA binding proteins associated with neuronal inclusions in amyotrophic lateral sclerosis (ALS) patients. Other neurodegenerative diseases are also characterized by neuronal protein aggregates, e.g. Huntington's disease, associated with polyglutamine (polyQ) expansions in the protein huntingtin. Here we discuss our recent paper establishing similarities between aggregates of TDP-43 that have short glutamine and asparagine (Q/N)-rich modules and are soluble in detergents, with those of polyQ and PIN4C that have large Q/N-rich domains and are detergent-insoluble. We also present new, similar data for FUS. Together, we show that like overexpression of polyQ or PIN4C, overexpression of FUS or TDP-43 causes inhibition of the ubiquitin proteasome system (UPS) and toxicity, both of which are mitigated by overexpression of the Hsp40 chaperone Sis1. Also, in all cases toxicity is enhanced by the [PIN+] prion. In addition, we show that the Sis1 mammalian homolog DNAJBI reduces toxicity arising from overexpressed FUS and TDP-43 respectively in human embryonic kidney cells and primary rodent neurons. The common properties of these proteins suggest that heterologous aggregates may enhance the toxicity of a variety of disease-related aggregating proteins, and further that chaperones and the UPS may be key therapeutic targets for diseases characterized by protein inclusions.

Overexpression of the essential Sis1 chaperone reduces TDP-43 effects on toxicity and proteolysis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by selective loss of motor neurons with inclusions frequently containing the RNA/DNA binding protein TDP-43. Using a yeast model of ALS exhibiting TDP-43 dependent toxicity, we now show that TDP-43 overexpression dramatically alters cell shape and reduces ubiquitin dependent proteolysis of a reporter construct. Furthermore, we show that an excess of the Hsp40 chaperone, Sis1, reduced TDP-43's effect on toxicity, cell shape and proteolysis. The strength of these effects was influenced by the presence of the endogenous yeast prion, [PIN+]. Although overexpression of Sis1 altered the TDP-43 aggregation pattern, we did not detect physical association of Sis1 with TDP-43, suggesting the possibility of indirect effects on TDP-43 aggregation. Furthermore, overexpression of the mammalian Sis1 homologue, DNAJB1, relieves TDP-43 mediated toxicity in primary rodent cortical neurons, suggesting that Sis1 and its homologues may have neuroprotective effects in ALS.

Heterologous aggregates promote de novo prion appearance via more than one mechanism.

Prions are self-perpetuating conformational variants of particular proteins. In yeast, prions cause heritable phenotypic traits. Most known yeast prions contain a glutamine (Q)/asparagine (N)-rich region in their prion domains. [PSI+], the prion form of Sup35, appears de novo at dramatically enhanced rates following transient overproduction of Sup35 in the presence of [PIN+], the prion form of Rnq1. Here, we establish the temporal de novo appearance of Sup35 aggregates during such overexpression in relation to other cellular proteins. Fluorescently-labeled Sup35 initially forms one or a few dots when overexpressed in [PIN+] cells. One of the dots is perivacuolar, colocalizes with the aggregated Rnq1 dot and grows into peripheral rings/lines, some of which also colocalize with Rnq1. Sup35 dots that are not near the vacuole do not always colocalize with Rnq1 and disappear by the time rings start to grow. Bimolecular fluorescence complementation failed to detect any interaction between Sup35-VN and Rnq1-VC in [PSI+][PIN+] cells. In contrast, all Sup35 aggregates, whether newly induced or in established [PSI+], completely colocalize with the molecular chaperones Hsp104, Sis1, Ssa1 and eukaryotic release factor Sup45. In the absence of [PIN+], overexpressed aggregating proteins such as the Q/N-rich Pin4C or the non-Q/N-rich Mod5 can also promote the de novo appearance of [PSI+]. Similar to Rnq1, overexpressed Pin4C transiently colocalizes with newly appearing Sup35 aggregates. However, no interaction was detected between Mod5 and Sup35 during [PSI+] induction in the absence of [PIN+]. While the colocalization of Sup35 and aggregates of Rnq1 or Pin4C are consistent with the model that the heterologous aggregates cross-seed the de novo appearance of [PSI+], the lack of interaction between Mod5 and Sup35 leaves open the possibility of other mechanisms. We also show that Hsp104 is required in the de novo appearance of [PSI+] aggregates in a [PIN+]-independent pathway.

The yeast Batten disease orthologue Btn1 controls endosome-Golgi retrograde transport via SNARE assembly.

The human Batten disease gene CLN3 and yeast orthologue BTN1 encode proteins of unclear function. We show that the loss of BTN1 phenocopies that of BTN2, which encodes a retromer accessory protein involved in the retrieval of specific cargo from late endosomes (LEs) to the Golgi. However, Btn1 localizes to Golgi and regulates soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor (SNARE) function to control retrograde transport. Specifically, BTN1 overexpression and deletion have opposing effects on phosphorylation of the Sed5 target membrane SNARE, on Golgi SNARE assembly, and on Golgi integrity. Although Btn1 does not interact physically with SNAREs, it regulates Sed5 phosphorylation by modulating Yck3, a palmitoylated endosomal kinase. This may involve modification of the Yck3 lipid anchor, as substitution with a transmembrane domain suppresses the deletion of BTN1 and restores trafficking. Correspondingly, deletion of YCK3 mimics that of BTN1 or BTN2 with respect to LE-Golgi retrieval. Thus, Btn1 controls retrograde sorting by regulating SNARE phosphorylation and assembly, a process that may be adversely affected in Batten Disease patients.

Btn3 is a negative regulator of Btn2-mediated endosomal protein trafficking and prion curing in yeast.

Yeast Btn2 facilitates the retrieval of specific proteins from late endosomes (LEs) to the Golgi, a process that may be adversely affected in Batten disease patients. We isolated the putative yeast orthologue of a human complex I deficiency gene, designated here as BTN3, as encoding a Btn2-interacting protein and negative regulator. First, yeast overexpressing BTN3 phenocopy the deletion of BTN2 and mislocalize certain trans-Golgi proteins, like Kex2 and Yif1, to the LE and vacuole, respectively. In contrast, the deletion of BTN3 results in a tighter pattern of protein localization to the Golgi. Second, BTN3 overexpression alters Btn2 localization from the IPOD compartment, which correlates with a sharp reduction in Btn2-mediated [URE3] prion curing. Third, Btn3 and the Snc1 v-SNARE compete for the same binding domain on Btn2, and this competition controls Btn2 localization and function. The inhibitory effects upon protein retrieval and prion curing suggest that Btn3 sequesters Btn2 away from its substrates, thus down-regulating protein trafficking and aggregation. Therefore Btn3 is a novel negative regulator of intracellular protein sorting, which may be of importance in the onset of complex I deficiency and Batten disease in humans.

RNAi mediated silencing of a wall associated kinase, OsiWAK1 in Oryza sativa results in impaired root development and sterility due to anther indehiscence: Wall Associated Kinases from Oryza sativa.

The Wall-Associated Kinase, one of the receptor-like kinase (RLK) gene families in plant, plays important roles in cell expansion, pathogen resistance and heavy metal stress tolerance in Arabidopsis thaliana. Here, we isolated a cDNA encoding a novel WAK from indica rice and designated as OsiWAK1 (Oryza sativa indica WAK-1). In this study, the RNAi construct with OsiWAK1 gene cloned in sense and antisense orientation separated by a functional intron under constitutive promoter, was introduced through biolistic gene gun method into the rice cultivar "IR-50" to determine the effect of OsiWAK1 transcript silencing on rice plant development. Examination of the transgenic plants reveals that OsiWAK1 transcript silencing in rice results in dwarf plants because of the reduction in the size of leaves, flag-leaves, internodes and panicle. The development of root primordia during germination, root hairs and lateral rooting was also effected. Microscopic analysis revealed that the decrease in size is due to reduction in the cell size but not the number of cells. In addition, the transgenic plants also exhibited sterile phenotype due to anther indehiscence and 40 % reduction in pollen viability. These data suggest that OsiWAK1 may play an important role in rice plant growth and development.

Isolation and Expression analysis of OsPME1, encoding for a putative Pectin Methyl Esterase from Oryza sativa (subsp. indica).

Pectin Methyl Esterases (PMEs) play an essential role during plant development by affecting the mechanical properties of the plant cell walls. Recent studies indicated that PMEs play important role in pollen tube development. In this study, we isolated a 1.3 kb cDNA clone from rice panicle cDNA library. It contained a 1038 bp of open reading frame (ORF) encoding for a putative pectin methyl esterase of 345 aminoacids with a 20 aminoacid signal peptide and was hence designated as OsPME1 (Oryza sativaPectin Methyl Esterase 1). It contained the structural arrangement GXYXE and GXXDFIF, found in the active groups of all PMEs. OsPME1 gene product shared varying identities, ranging from 52 % to 33 % with PMEs from other plant species belonging to Brassicaceae, Fabaceae, Amaranthaceae and Funariaceae. Southern blot analysis indicated that PME1 exists as a single copy in the rice genome. Expression pattern analysis revealed that OsPME1 is expressed only in pollen grains, during the later stages of their development and was also regulated by various abiotic stress treatments and phytohormones. Functional characterization of this pollen specific PME from rice would enable us to understand its role in pollen development.

Overexpression of OsiSAP8, a member of stress associated protein (SAP) gene family of rice confers tolerance to salt, drought and cold stress in transgenic tobacco and rice.

We describe here the isolation and characterization of OsiSAP8, a member of stress Associated protein (SAP) gene family from rice characterized by the presence of A20 and AN1 type Zinc finger domains. OsiSAP8 is a multiple stress inducible gene, induced by various stresses, namely heat, cold, salt, desiccation, submergence, wounding, heavy metals as well as stress hormone Abscisic acid. OsiSAP8 protein fused to GFP was localized towards the periphery of the cells in the epidermal cells of infiltrated Nicotiana benthamiana leaves. Yeast two hybrid analysis revealed that A20 and AN1 type zinc-finger domains of OsiSAP8 interact with each other. Overexpression of the gene in both transgenic tobacco and rice conferred tolerance to salt, drought and cold stress at seed germination/seedling stage as reflected by percentage of germination and gain in fresh weight after stress recovery. Transgenic rice plants were tolerant to salt and drought during anthesis stage without any yield penalty as compared to unstressed transgenic plants.

Wall associated kinases from plants - an overview.

Wall Associated kinases (WAKs) represent a unique class of receptor-like kinase genes that span the plasma membrane and allow cells to recognize and respond to their extracellular environment 26 WAK/WAK-like genes were identified from the Arabidopsis genome. Functional studies of the different WAK members in Arabidopsis demonstrated that they are involved in various functions in plants, including pathogen resistance, heavy-metal tolerance and plant development. 125 genes from rice (subsp. Japonica) belonging to wall associated kinase gene family were identified by reiterative database searches. We isolated a new member of WAKs in rice, designated as OsiWAK1, the silencing of which led to impaired root development and sterility due to anther indehiscence. In the current review, we discuss about the isolation and identification of WAK members from various plant species, different domains found in the WAK proteins that make them unique and the various roles played by WAKs in the plant growth and development.