N-terminal region of Saccharomyces cerevisiae eRF3 is essential for the functioning of the eRF1/eRF3 complex beyond translation termination.

BACKGROUND: Termination of translation in eukaryotes requires two release factors, eRF1, which recognizes all three nonsense codons and facilitates release of the nascent polypeptide chain, and eRF3 stimulating translation termination in a GTP-depended manner. eRF3 from different organisms possess a highly conservative C region (eRF3C), which is responsible for the function in translation termination, and almost always contain the N-terminal extension, which is inessential and vary both in structure and length. In the yeast Saccharomyces cerevisiae the N-terminal region of eRF3 is responsible for conversion of this protein into the aggregated and functionally inactive prion form. RESULTS: Here, we examined functional importance of the N-terminal region of a non-prion form of yeast eRF3. The screen for mutations which are lethal in combination with the SUP35-C allele encoding eRF3C revealed the sup45 mutations which alter the N-terminal domain of eRF1 and increase nonsense codon readthrough. However, further analysis showed that synthetic lethality was not caused by the increased levels of nonsense codon readthrough. Dominant mutations in SUP35-C were obtained and characterized, which remove its synthetic lethality with the identified sup45 mutations, thus indicating that synthetic lethality was not due to a disruption of interaction with proteins that bind to this eRF3 region. CONCLUSION: These and other data demonstrate that the N-terminal region of eRF3 is involved both in modulation of the efficiency of translation termination and functioning of the eRF1/eRF3 complex outside of translation termination.

Correct GPI-anchor synthesis is required for the incorporation of endoglucanase/glucanosyltransferase Bgl2p into the Saccharomyces cerevisiae cell wall.

The SSU21/MCD4 gene encodes an essential component of the glycosylphosphatidylinositol (GPI)-anchor synthesis pathway in Saccharomyces cerevisiae. Here we demonstrate that the ssu21 mutation affected the transport and the incorporation into the cell wall of the major non-GPI yeast cross-linker - endoglucanase/glucanosyltransferase Bgl2p. This mutation also led to a decrease in the levels of both known types of cell wall mannoproteins, those covalently linked with glucan and SDS-extractable proteins. Our results indicate that the precision of the GPI-anchor synthesis is essential for cell wall assembly and suggest the strong interdependence of different groups of cell wall proteins during their incorporation into the cell wall.

C-terminal truncation of alpha-COP affects functioning of secretory organelles and calcium homeostasis in Hansenula polymorpha.

In eukaryotic cells, COPI vesicles retrieve resident proteins to the endoplasmic reticulum and mediate intra-Golgi transport. Here, we studied the Hansenula polymorpha homologue of the Saccharomyces cerevisiae RET1 gene, encoding alpha-COP, a subunit of the COPI protein complex. H. polymorpha ret1 mutants, which expressed truncated alpha-COP lacking more than 300 C-terminal amino acids, manifested an enhanced ability to secrete human urokinase-type plasminogen activator (uPA) and an inability to grow with a shortage of Ca2+ ions, whereas a lack of alpha-COP expression was lethal. The alpha-COP defect also caused alteration of intracellular transport of the glycosylphosphatidylinositol-anchored protein Gas1p, secretion of abnormal uPA forms, and reductions in the levels of Pmr1p, a Golgi Ca2+-ATPase. Overexpression of Pmr1p suppressed some ret1 mutant phenotypes, namely, Ca2+ dependence and enhanced uPA secretion. The role of COPI-dependent vesicular transport in cellular Ca2+ homeostasis is discussed.