No abstract

NUF1/SPC110, encoding a nuclear filament‐related protein which is a component of the yeast spindle pole body (SPB), has been identified in a screen designed to isolate genes encoding targets of yeast calmodulin. Spc110p interacts with calmodulin by two different criteria and the calmodulin interacting region has been localized within the C‐terminus of the protein. Point mutations between residues 898 and 917 further define the calmodulin binding site within this region. Mutations in this domain which abolish calmodulin binding in vitro prevent Spc110p function in vivo, demonstrating that calmodulin binding by Spc110p has important functional consequences. In keeping with a role for calmodulin in Spc110p function, we show that calmodulin localizes to the yeast SPB when cells are prepared under appropriate conditions. Non‐functional mutant Spc110 proteins which cannot bind calmodulin are present at lowered steady‐state levels in the cell; when their level is increased by elevated gene dosage, partial recovery of Spc110p function is seen. Overexpression of calmodulin suppresses the defect(s) associated with the mutant Spc110 proteins, supporting the notion that Spc110p stability is a consequence of its ability to bind calmodulin and pointing to a direct role for calmodulin in Spc110p function.

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The proU loci of Salmonella typhimurium and Escherichia coli encode high-affinity glycine betaine transport systems which play an important role in survival under osmotic stress. Transcription of the proU locus is tightly regulated by osmolarity and this regulation appears to be mediated by osmotically induced changes in DNA supercoiling. In order to study the regulatory mechanisms involved we have cloned and characterized the proU locus of S. typhimurium by an in vivo transductional procedure. The locus is shown to consist of at least three genes, designated proVWX, cotranscribed as a single operon. The first gene in the operon encodes a protein sharing considerable sequence identity with ATP-binding proteins from other periplasmic transport systems. Unexpectedly, the highly expressed periplasmic glycine betaine binding protein was found to be encoded by a distal gene, proX, in the operon. The operon has no significant internal promoters but is expressed from a single osmoregulated promoter whose transcription start site has been mapped. The proU promoter of E. coli has also been sequenced and the transcription start site shown to be similar to that of S. typhimurium. Evidence is presented which suggests that, besides de novo glycine betaine uptake, an important function of ProU may be the recapture and recycling of other osmolytes that leak from the cell.

A novel gene fusion approach which may be of more general use has been developed for investigating the function of calmodulin in the budding yeast Saccharomyces cerevisiae. By fusing a portion of the Staphylococcus aureus spa gene (encoding protein A) to CMD1, the S. cerevisiae gene encoding calmodulin, we have generated a yeast calmodulin with an affinity tag able to bind immunoglobulins. The chimaeric protein A-calmodulin (ProtA-CaM) polypeptide functions in vivo and shows Ca(2+)-dependent binding to calmodulin target proteins. The spa-CMD1 fusion has been used (i) to prepare (by affinity chromatography) a fraction of yeast proteins which interact with calmodulin, (ii) to isolate genes encoding calmodulin target proteins by direct screening of an expression library, and (iii) to visualize calmodulin-binding proteins in crude extracts by Western blot analysis.