Article 6

Normington, K., K. Kohno, Y. Kozutsumi, M-J. Gething, & J. Sambrook (1989) S. cerevisiae encodes an essential protein homologous in sequence and function to mammalian BiP. Cell 57: 1223-1236.

BiP is an ER localized member of the HSP70 (heat shock protein 70) family. Studies of BiP function in mammalian cells showed that it bound transiently to a number of nascent secreted proteins and strongly to improperly folded proteins blocking their exit from the ER. As such, it was hypothesized that BiP played a very important, possibly essential cellular function and would likely be evolutionarily conserved. The mammalian BiP gene had been cloned, and this article describes the use of this gene to identify the yeast BiP gene.

Table 1 describes the results of quantitative mating assays between strains carrying mutations in KAR2. The assay is described in detail in Rose & Fink (1987). Matings involving kar2 mutants are defective in nuclear fusion, karyogamy (see Chapter 3). The cytoplasms of the mating partners fuse and cytoplasmic organelles such as the mitochondria become distributed throughout the common cytoplasm. However, the resulting zygote contains two haploid nuclei and cell division results in haploid products containing one or the other of these two nuclei.

In a quantitative mating assay, cytoplasmic fusion is monitored by following the status of the mitochondrial DNA (mtDNA). A [rho+] strain contains wild-type mtDNA, has functional mitochondria, and forms large (grande) creamy-colored colonies on YPD media. A [rho-] strain has lost its mtDNA, lacks functional mitochondria, and forms small (petite) colonies on YPD because it cannot utilize the products of glucose fermentation. Ethidium bromide interferes with mtDNA replication and growth in ethidium bromide is commonly used to produce [rho-] strains. In addition to colony size, one can distinguish [rho-] from [rho+] by its interaction with the ade2 mutation. An ade2 [rho-1-] strain is pink in color while an ade2 [rho-] strain is white.

The genotypes of the strains used in a typical quantitative mating assay are given below:

MATa kar2 ade2 LYS2 [rho-] x MAT KAR2 ADE2 lys2 [rho+] white colonies pink colonies

If mating is complete, both nuclear and cytoplasmic fusion occurs and diploid ade+ lys+ prototrophs will be produced that form creamy-white colonies on YPD. If only cytoplasmic fusion occurs, no prototrophs will be produced and the process is referred to as cytoduction. When these zygotes divide by mitosis they produce some MATa kar2 ade2 LYS2 [rho+] progeny, called cytoductants, which are pink. In a typical mating such as the one shown above nuclear fusion is usually not 100% defective but is only partially defective. This is quantified by calculating the ratio of cytoductants to prototrophs resulting from the mating. The number of cytoductants is determined by the number of pink colonies and the number of prototrophs is determined by the number of colonies growing on the appropriate selective medium. In the case shown above this would be SM lacking adenine and lysine.

1. The authors tested a number of hybridization conditions using Southern analysis to adjust the stringency at which they would screen their library. They settled on conditions that detected one strongly hybridizing fragment plus several weakly hybridizing fragments.

(a) What were these conditions?

(b) Why were they not able to use the typical high stringency conditions?

(c) What did they hope was the single strongly hybridizing fragment? The several weak fragments?

2. The yeast BiP gene was initially isolated from a cDNA library.

(a) Describe a cDNA library. Why is this type of library usually not used for Saccharomyces studies?

(b) Describe the first probe used to screen the library.

(c) Two hundred potential BiP gene clones were obtained from the initial screening of 20 000 lambda clones. These were screened at high stringency with a second probe. Describe the probe and the reason for doing this second screen.

(d) Of the 20 clones that passed the second screen, 12 were sequenced and only one was novel. What were the other 11 and why were these not eliminated by the second screen? Other than sequencing, how might these 11 clones be excluded and found to contain other known HSP70 genes?

(e) How was a full-length copy of the yeast BiP gene obtained (Figure 2C)?

3. What sequence features of yeast BiP suggest that it is an ER localized protein and thus differs significantly from its close homologues Ssblp and the other HSP70 proteins that are found in the cytoplasm?

4. What does it mean when the authors say that yeast BiP exhibits 67% identity and 84% similarity to mammalian BiP?

5. How did the authors determine that their yeast BiP gene was identical to KAR21 What is KAR21

6. Describe how the authors demonstrated that KAR2 is an essential gene.

7. Describe the experiment used to demonstrate that the N-terminal 42 residues of yeast BiP function as a signal sequence to localize BiP to the lumen of the ER.

8. Diagram the mammalian BiP expression constructs in plasmids YEpCup-MB and YEpCup-FusB.

(a) Why is the CUP1 promoter used?

(b) How do the protein products of these two constructs differ?

9. Strains carrying mutations in KAR2 are temperature sensitive for growth.

(a) Does mammalian BiP complement the ts phenotype of kar2-ll

(b) Does it complement as well as the cloned yeast BiP gene and what is the evidence?

10. Strains carrying mutations in KAR2 exhibit a defect in karyogamy; that is, mating cells fuse their cytoplasms but not their nucleii. True diploids are not formed but mitochondria are exchanged.

(a) Diagram the cross from Table 1 between W303-2/none and SEY6210. [Note that there is a typo in the table legend. SEY6210 is a 'MATa tester strain.]

(b) What do the numbers presented in Table 1 represent?

(c) Strains W303-2 and KNH2/YEpCup-YB are positive controls in this experiment but differ. Discuss.

(d) Strain KNH2/YEpCup is a negative control. Discuss.

(e) What data demonstrate that reduced expression of yeast BiP only partially complements kar2-l?

(f) What data demonstrate that expression of mammalian BiP partially complements kar2-ll

11. What is the significance of showing that mammalian BiP complements kar2-ll

12. What unique features (sequence, expression, localization, etc.) are shared by

Kar2p and mammalian BiP that distinguish them from the other HSP70

proteins?

REFERENCE

Rose, M. & G. Fink (1987) KAR1, a gene required for function of both intranuclear and extranuclear microtubules in yeast. Cell 48: 1047-1060.

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