Arp2p, a potential substrate for NatB acetyltransferase in fission yeast, is important in actin patch nucleation and motility in arp2-1 and arm1 ts mutants

The actin cytoskeleton is a dynamic and complex structure in fission yeast that plays a major function in many cell processes including cellular growth, septa formation, endocytosis and cellular division. Computational studies have shown that Arp2p, which forms part of the Arp2/3 complex, is a potential substrate of NatB acetyltransferase which has specificity for proteins possessing an N-terminal Met-Asp or Met-Glu sequence motif. In arm1- mutants the loss of function of Arm1p, an auxillary subunit required for NatB activity, results in a temperature sensitive phenotype characterized by multiple septa, failure of endocytosis, and the inability to form actin cables. A temperature sensitive mutant of Schizosaccharomyces pombe arp2 gene exhibits a similar phenotype as seen by the formation of improper septa, slow growth, and the delocalization of actin patches. Four expression vectors encoding the open reading frames of arp2 and cdc8 (tropomyosin) were constructed with a modification changing the second residue to a Histidine, believed to mimic the charge distribution of natural acetylation by NatB. Constructs tested in normal yeast strains remained viable and grew normally in the presence of Met-His Arp2p and tropomyosin. Analysis of their ability to suppress the mutant phenotypes of arp2-1 and arm1- mutants is an area of research to be explored in future studies.

Two-hybrid analysis of the interaction between the fission yeast proteins Sal3 and Cdc25

Cdc25 is a mitosis triggering phosphatase in Schizosaccharomyces pombe, and is transported in to the nucleus during G2 phase by the importin-β protein Sal3. Cdc25 triggers mitosis and cell division by dephosphorylating tyrosine 15 of Cdc2. In sal3 mutants, Cdc25 is not transported into the nucleus and the cells halt in G2. The purpose of this study is to use a two-hybrid system to determine the nature of the relationship between Sal3 and Cdc25. Previous research has failed to detect any interaction between the two proteins, but specific modifications were made to the two-hybrid system in this study including the separation of Sal3 into its two binding domains, the addition of fluorescent tags to the fusion protein, and the reversal of plasmids in the fusion proteins. Unique PCR primers were successfully designed, based on a multiple alignment of Sal3 and its homologues, to separate Sal3 into its two domains.

The infuence of yeast rhomboid protease on the import of Tic40 into the yeast mitochondrion

Yeast rhomboid protease (Rbd1p) was found to act in the processing of Tic40 components in the yeast mitochondrion. Rhomboid protease was shown to have effects on the number of different Tic40 configurations displayed, the ratio of different configurations to one another and the targeting of Tic40 configurations within the yeast mitochondrion. The effects of Rbd1p on the ratio and targeting of different Tic40 configurations were also found to be dependent on the developmental stage of the yeast. Tic40 deletion constructs were expressed in yeast strains with active yeast rhomboid protease and in corresponding strains lacking Rbd1p. The processing of Tic40 differed between deletion constructs and between strains with and without yeast rhomboid protease. This indicates that rhomboid protease can affect the processing of Tic40 and the sequence of Tic40 can affect the activity of rhomboid protease with respect to Tic40. Tic40 is suspected to be involved in the regulation of plastid protein import. Rhomboid protease is shown here to affect the properties of Tic40 which have made it a candidate for a regulator of plastid protein import.