Sadler, I., A. Chiang, T. Kurhara, J. Rothblatt, J. Way, & P. Silver (1989) A yeast gene important for protein assembly into the endoplasmic reticulum and the nucleus has homology to DnaJ, an Escherichia coli heat shock protein. J. Cell Biol. 109: 2665-2675.
This article describes the cloning of yeast SEC63. It also illustrates how the same gene can be identified in different selection/screens that use different phenotypes as the basis of the selection/screen. The authors had developed a selection method for mutations in genes involved in nuclear protein localization. They constructed a gene encoding a hybrid cytochrome cl protein fused in-frame to the nuclear localization signal (NLS) from Gal4 protein or the SV40 T antigen, and this fusion was expressed from the high-level constitutive AD HI promoter. Cytochrome cl is encoded by CYC1, synthesized by cytoplasmic ribosomes, and post-translationally enters the mitochondrion for functions in the electron transport chain. Mutants in CYC1 are unable to grow on nonfermentable carbon sources. Gal4p is the transcription activator of the GAL genes and is normally found in the nucleus. SV40 T antigen is also a nuclear protein.
The NLS cytochrome cl fusion protein localized to the nucleus was so abundantly expressed that it was toxic. The authors reasoned that mutants with alterations in the nuclear transport system might be resistant to the toxic effects of the fusion protein and also might allow some entry into the mitochondrion by default. As a result these mutant strains would be able to grow on a nonfermentable carbon source. Mutants were selected based on their ability to grow on glycerol at 30°C where partially functional mutant proteins could lead to reduced rates of nuclear localization. Since nuclear localization is an essential function the mutants were secondarily screened for temperature-sensitive growth at 37°C where the altered product would be completely nonfunctional. Mutations in three so-called NPL genes were isolated.
1. The immunofluorescence studies of the NLS cytochrome cl fusion proteins suggested that they were found 'at the nucleus and opposed to the nuclear envelope'. Based on this, the authors suggested that the npl mutant genes could be involved in the early stages of ER localization. This was tested and NPL1 was found to be SEC63.
(a) Diagram the cross showing that npll-1 and sec63-l are alleles. Give the genotype and phenotype of the parent strains, the diploid, and the results of tetrad analysis.
(b) What phenotype was assayed in the tetrads?
2. NPL1 was cloned by complementation of the ts-lethal phenotype of npll-1.
(a) Describe the library.
(b) Give the genotype of the host strain.
(c) What phenotype was used to select for transformants carrying a library plasmid?
(d) What phenotype was used to screen transformants for those potentially carrying the NPL1 allele?
(e) Why is it essential to recover the plasmid from these transformants and reintroduce it into the npll-1 strain?
3. Deletion analysis of plasmid pTKl localized NPL1 within the 6 kbp yeast insert. Describe how this was done.
4. Targeted integration was used to demonstrate that the cloned complementing fragments truly contained NPL1/SEC63 and not a suppressor gene.
(a) Describe what was done. Be sure to include a diagram of the integrating plasmid and diagram the cross to the npll-1 strain.
(b) Why was this process repeated with a sec63-l strain?
5. Describe the method used to demonstrate that NPL1ISEC63 is essential.
6. Do npll-1, npll-2, and sec63-l exhibit a similar phenotype with regard to CPY maturation? Are these null alleles? Discuss the significance with regard to the finding that SEC63 is an essential gene.
7. Does sec63-l exhibit an npl phenotype?
8. Discuss the following 'striking' sequence features of Sec63 protein, (a) The three potential membrane-spanning domains.
(b) The homology to the DnaJ protein of E. coli.
(c) The C-terminal 52 residues.
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