![Fig. 2. The main pathway of GPI biosynthesis in yeast. Since no GPI intermediates are detectable in wild type cells, a likely pathway is proposed based on structures of GPIs accumulating in well-characterized gpi mutants [62,68,71,72,166]. Biosynthetic steps are numbered in accordance with the description in Table 1. CP2 is the most polar lipid accumulating in gpi8mutants. M4 is also transferred to GPI proteins, since GPI7 is non-essential [71]. All genes in the pathway encode integral membrane proteins having at least one and in most cases multiple membrane spanning domains.](/figures/fig-2-the-main-pathway-of-gpi-biosynthesis-in-yeast-since-no-2d6zj2vg.webp)
Q2. What is the role of GPI proteins in the cell wall?
Other GPI proteins may act as enzymes to make and break glycosidic linkages and are required for elaboration of the cell wall and its reshaping during bud emergence, cell separation, mating or entry into stationary phase.
Q3. How did the GPI7 dependent targeting to the septum work?
GPI7 dependent targeting to the septum could be conveyed to Eng1p by attaching the GPI anchor attachment signal plus 27 amino acids of theωminus region of EGT2 to the C-terminal end of ENG1.
Q4. What are the common amino acids used as anchors in yeast?
While several amino acids can serve as anchor attachment sites in other organisms (Ser, Asp, Ala, Asn, Gly, Cys), only Asn and Gly have been found so far in yeast.
Q5. How many genes are needed for the addition of GPI anchors?
after 20 years of intensive work, genes required for the addition of about everyone of the different structural elements of the GPI anchor have been identified, but new subunits and regulatory elements of the identified enzymes continue to be discovered.
Q6. What is the quality of bioinformatics predictors of GPI proteins?
the quality of bioinformatics predictors of GPI proteins relies on the quality and amount of biochemical experiments establishing the presence of GPI anchors on the proteins used as the training set [7].
Q7. How do you determine the location of GPI proteins in the cell wall?
Many GPI proteins are transcribed in a cell cycle dependent manner and their location in the cell wall seems to be determined by the polarization of the secretory pathway at the time of their expression (reviewed by Smits et al. [27]).
Q8. How can the authors obtain biochemical evidence of GPI anchoring?
Biochemical evidence can be obtained through indirect methods, e.g., through the demonstration of a loss of hydrophobicity upon treatment with PI-specific phospholipase C (PI-PLC), through metabolic labeling with anchor components such as [3H] inositol, by the loss of surface localization upon site directed mutation of a putativeω site, or by the demonstration of a covalent, alkali-resistant, glucanase-sensitive association with the cell wall, for which the addition of a GPI anchor is a prerequisite.
Q9. How does the GPI7p protein transfer from the ER?
Apart from exhibiting a deficiency in the EtN-P transfer onto Man2 detected in vivo and in vitro, gpi7Δ mutants transport GPI proteins from the ER to the Golgi more slowly than wild type.
Q10. Why was Gpi7p claimed to be present in a high molecular weight?
The bulk of Gpi7p was claimed to be present in a high molecular weight complex at the plasma membrane, because treatment of spheroplasts with protease eliminated the high molecular weight form of Gpi7p [71].
Q11. What is the role of EtN-P in the ER to Golgi transport?
Recent data indicate that the EtN-P group added by Mcd4p is required for the recognition of GPIs by Gpi10p as well as by the transamidase complex and that this EtN-P group plays a role in ER to Golgi transport of GPI proteins and also in ceramide remodeling [65].
Q12. How can the authors remove GPI-CWPs from the cell walls?
GPI-CWPs can be selectively removed from the cell walls by treatment with endo-β1,6-endoglucanase or hydrofluoric acid (HF) [22].
Q13. How many genes have been found in other eukaryotes?
Almost all the genes identified as being essential for a given enzymatic step in mammalian or yeast cells have found their homologues in other eukaryotes.
Q14. What is the homology between yeast and mammalian genes?
Although the homology between yeast an mammalian genes is usually low, it tends to be higher for those proteins, which have a catalytic activity (Table 1).
Q15. What is the role of the transamidase complex in the ER?
Genetic and biochemical approaches have so far identified 5 proteins that encode the subunits of the transamidase complex, which transfers the GPI lipid to the newly made GPI protein in the ER (step 11, Fig. 2 and Table 1) [78–82].