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Abstract: Class A heat shock factors (Hsfs) of Arabidopsis are known to function as transcriptional activators of stress genes. Genetic and functional analysis suggests that HsfA1a and HsfA1b are central regulators required in the early phase of the heat shock response, which have the capacity to functionally replace each other. In order to examine Hsf interaction in vivo, we conducted interaction assays using bimolecular fluorescence complementation (BiFC) on Arabidopsis protoplasts co-transformed with suitable Hsf-YFP fusion genes. BiFC assays were quantified with confocal laser scanning microscopy and flow cytometry, and confirmed with immunoprecipitation assays. For each Hsf we could not only demonstrate homomeric interactions but also detect heteromeric interaction between HsfA1a and HsfA1b. Truncated versions of these of Hsfs, containing deletions of the oligomerization domains (ODs), provided clear evidence that the ODs are required and sufficient for the HSF interaction in vivo. By contrast there was only homomeric but no heteromeric interaction detected between two different class B Hsf transcription factors (HsfB1 and HsfB2b) in a yeast two-hybrid assay. HsfB1/HsfB2b functions are not directly linked with the expression of conventional heat shock genes; class B Hsfs are devoid of the activation domain motif conserved in class A Hsfs. In order to identify other proteins interacting with HsfB1 and HsfB2b we performed yeast two-hybrid screenings of cDNA libraries. Three of the identified proteins were common to both screenings. This suggests that HsfB1 and HsfB2b may be involved in complex regulatory networks, which are linked to other stress responses and signaling processes. [Copyright &y& Elsevier]

Abstract: KRIT1 is a disease gene responsible for Cerebral Cavernous Malformations (CCM). It encodes for a protein containing distinct protein–protein interaction domains, including three NPXY/F motifs and a FERM domain. Previously, we isolated KRIT1B, an isoform characterized by the alternative splicing of the 15th coding exon and suspected to cause CCM when abnormally expressed. Combining homology modeling and docking methods of protein-structure and ligand binding prediction with the yeast two-hybrid assay of in vivo protein–protein interaction and cellular biology analyses we identified both structural and functional differences between KRIT1A and KRIT1B isoforms. We found that the 15th exon encodes for the distal β-sheet of the F3/PTB-like subdomain of KRIT1A FERM domain, demonstrating that KRIT1B is devoid of a functional PTB binding pocket. As major functional consequence, KRIT1B is unable to bind Rap1A, while the FERM domain of KRIT1A is even sufficient for this function. Furthermore, we found that a functional PTB subdomain enables the nucleocytoplasmic shuttling of KRIT1A, while its alteration confers a restricted cytoplasmic localization and a dominant negative role to KRIT1B. Importantly, we also demonstrated that KRIT1A, but not KRIT1B, may adopt a closed conformation through an intramolecular interaction involving the third NPXY/F motif at the N-terminus and the PTB subdomain of the FERM domain, and proposed a mechanism whereby an open/closed conformation switch regulates KRIT1A nuclear translocation and interaction with Rap1A in a mutually exclusive manner. As most mutations found in CCM patients affect the KRIT1 FERM domain, the new insights into the structure–function relationship of this domain may constitute a useful framework for understanding molecular mechanisms underlying CCM pathogenesis. [Copyright &y& Elsevier]

Abstract: Animals with immune systems have two types of proteasomes, “standard proteasomes” and “immunoproteasomes” that respectively contain constitutively expressed catalytic subunits or interferon-γ-inducible catalytic subunits. Interestingly, proteasome assembly is biased against formation of most mixed proteasomes containing combinations of standard subunits and immunosubunits. We previously demonstrated that catalytic subunit propeptide differences contribute to this assembly specificity. In the current study, we investigated the contributions of catalytic subunit propeptides and C-terminal extensions to intra-proteasome protein–protein interactions that are potentially involved in mediating biased assembly of human proteasomes, and we found a number of interactions that differentially depended on these structures. For example, the C-terminal extension of standard subunit β2 is required for β2’s interaction with adjacent β3, whereas the C-terminal extension of immunosubunit β2i is dispensable for β2i’s interaction with β3. Taken together, our results suggest mechanisms whereby differential intra-proteasome interactions could contribute to proteasome assembly specificity. [Copyright &y& Elsevier]

Immunoproteasomes and standard proteasomes assemble by alternative pathways that bias against the formation of certain “mixed” proteasomes. Differences between β subunit propeptides contribute to assembly specificity and an assembly chaperone, proteassemblin, may be involved via differential propeptide interactions. We investigated possible mechanisms of biased proteasome assembly and the role of proteassemblin by identifying protein–protein interactions among human 20S proteasome subunits and proteassemblin using a yeast two-hybrid interaction assay. Forty-one interactions were detected, including five involving proteassemblin and contiguous β subunits, which suggests that proteassemblin binds to preproteasomes via a β subunit surface. Interaction between proteassemblin and β5, but not β5i, suggests that proteassemblin may be involved in the propeptide-dependent differential incorporation of these subunits. Interactions between proteassemblin and β1, β1i, and β7 suggest that proteassemblin may regulate preproteasome dimerization via interactions with the C-termini of these subunits, which in the mature 20S structure extend to contact opposing β subunit rings. [Copyright &y& Elsevier]

Homeodomain leucine zipper ( HDZip) genes encode putative transcription factors that are unique to plants. A function in regulating processes that are specific for plants is postulated, such as responses to environmental cues and developmental signals. This is supported by a growing body of evidence resulting from studies of HDZip genes in a variety of species. In addition to the previously isolated CPHB-1 and -2 genes, this paper reports the isolation of members of five families of Craterostigma plantagineum homeobox leucine zipper genes ( CPHB) via a yeast one-hybrid screening approach. Based on the sequence homology and protein interactions the encoded proteins (CPHB-3/4/5/6/7) were classified into HDZip class II and I genes. Homo- and heterodimerization of CPHB proteins within the same structurally related class has been demonstrated and the DNA-binding activity of CPHB proteins to two homeodomain recognition elements (HDE1 and HDE2) has been compared in yeast. All families of CPHB genes were modulated in their expression in response to dehydration in leaves and roots. CPHB-6 and CPHB-7 transcripts accumulated in leaves during early stages of dehydration and decreased after prolonged dehydration. Both transcripts were also induced in ABA-treated callus. CPHB-3/4/5 were down-regulated by dehydration in both leaves and roots. The results support the role of HDZips in regulating programs of gene expression in C. plantagineum that lead to desiccation tolerance. [ABSTRACT FROM AUTHOR]