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The movement proteins BC1 and BV1 of Abutilon mosaic geminivirus fused to glutathion-S-transferase (GST) and Flag-peptide were expressed in fission yeast (Schizosaccharomyces pombe) cells to analyse the fundamental intracellular distribution of these proteins in an eukaryotic cell in the absence of plant-specific factors. Most of BC1 protein sedimented rapidly after cell lysis and differential centrifugation. Using freeze-fracture immuno-labelling, the protein was detected in situ predominantly at plasma membranes and to a lower extent at cytoplasmic vesicles but not in the cytoplasm, the nuclei, or the mitochondria. Anti-BC1, anti-GST, and anti-Flag antibodies tagged smooth flecks only at the protoplasmic faces of the plasma membrane. The consequences of the BC1 behaviour for its use in two-hybrid analysis in yeast are discussed. In contrast, BV1 was detected mainly in the nucleus and partially in the cytoplasm but never associated with membranes.

The long terminal repeat (LTR) retrotransposons of the yeast Saccharomyces cerevisiae are similar in their structures and life cycles to animal retroviruses. The yeast LTR retrotransposon Ty3 does not transpose under conditions where the cellular stress response is activated. During stress, mature Ty3 proteins, indicative of the formation of intracellular Ty3 viruslike particles (VLPs), do not accumulate. In order to examine the role of stress proteins in Ty3 transposition, a sensitive genetic assay was developed to measure VLP formation. The assay employs a Ty3 element marked with a mutant allele of the yeast HIS3 gene (his3AI). To create a stable His+ phenotype, Ty3 must form VLPs, reverse transcribe Ty3 RNA into cDNA, and then insert the cDNA into either chromosomal or plasmid DNA. Using this assay, thermal inhibition of Ty3 transposition was evident at temperatures as low as 30 degrees C. The level of production of mature Ty3 proteins parallels the transposition frequency. Although overexpression of the yeast UBP3 gene allows VLPs to form and transposition to occur in the constitutively stressed ssa1 ssa2 strain, it does not alleviate the inhibition of these processes during stress induced by heat or ethanol. This suggests that the genetic and physical modes of stress response induction are not equivalent.

We have compared the functional and structural integrity of gap junction channels assembled from a Cx45 truncation mutant with those of gap junction channels assembled from wild-type (wt) Cx45 and Cx43. These channel-forming proteins are constitutively expressed in HeLa cells. The truncation mutant lacks the last 26 amino acids of the COOH-terminus, including nine serine phosphorylation sites that are associated with regulatory processes of these channels. We determined the presence of gap junction plaques in these cells with the immunogold freeze fracture technique, which showed that plaque formation is similar in all the clones investigated. Junctional permeability was probed with calcein transfer and flow cytometry analyses and junctional conductance was measured in cell pairs with double whole-cell patch-clamp techniques. For homotypic pairing only the truncated mutant did not form permeable channels. However, coupling was restored for heterotypic channels (pairing wtCx45- or wtCx43- with mutant-connexons), whose junctional communication was not different from that of the homotypic channels. Our results indicate that the presence of gap junction plaques does not warrant functional coupling and that heterotypic trCx45/wtCx45 channels can be regulated by the intact wtCx45 connexons. This dominant-positive effect is also operative when wtCx43 are paired with trCx45 connexons.