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Studies on siderophore uptake and pseudohyphal formation of Saccharomyces cerevisiae

  • 강창민 (대학원 생명과학과 분자생물학전공)

초록/요약

Iron is essential to all living organisms, and plays important roles in biological processes, such as enzymatic reactions, oxygen transport, and electron transport. Thus, most micro-organisms have developed various regulatory mechanisms for maintaining iron homeostasis. In S. cerevisiae, iron uptake is accomplished by reductive and siderophore-mediated iron uptake systems. Expression of the genes involved in the high-affinity iron uptake system is regulated by the transcriptional activator, Aft1. In this study, I examined the expression of two genes that are regulated by cellular iron concentrations: Sit1, a siderophore transporter, and Rck1, a MAPK-activated protein kinase. First, Sit1 regulation by Aft1 and transports siderophore for high-affinity iron uptake was studied. It has been reported that the physical interaction between Aft1 and Sit1 regulates ubiquitination of Sit1.In the presented study, the interaction between Sit1 and Aft1 induced stabilization of Sit 1 and its localization to the plasma membrane. An Msn5-deletion mutant strain and an S. cerevisiae strain transformed with the AFT1–1up plasmid, which results in Aft1 accumulation in the nucleus, showed lower ferrioxamine B(FOB) uptake activity. An Aft1 Y179F mutant strain, in which Aft1 failed to physically interact with Sit1, showed lower Sit1 protein stability and decreased FOB uptake activity. Additionally, strains treated with MG132 and PMSF, which are inhibitors of proteasomes and serine proteases, respectively, showed higher Sit1 protein levels compared to untreated strains. Altogether, these results suggest that the physical interaction between Aft1 and Sit1 is an important factor in the function of Sit1. Next, to determine the relationship between iron homeostasis and pseudohyphal formation by Rck1, MLY40/41 diploid strains were grown under nitrogen starvation conditions and various iron concentrations. Previous reports showed that Rck1 induced pseudohyphal growth in S. cerevisiae via MAPK and cAMP/PKA signaling, and that Rck1 is regulated by cellular iron concentrations. However, pseudohyphal formation was not regulated by cellular iron concentration. To explain the discrepancy, the focus of this study was to find other possible candidates, and found that Rck1 activated phosphorylation of the deubiquitinase Ubp3 through a direct protein interaction between Rck1 and Ubp3. The N-terminal Bre5 binding region of Ubp3 physically interacted with Rck1, and Ubp3 and Rck1 co-precipitated. Over-expression of Ubp3 using a high-copy plasmid resulted in the up-regulation of Ras2, and deletion of UBP3 blocked the up-regulation of Ras2 by Rck1 over-expression. Treatment with the proteasome inhibitor MG132 resulted in accumulation of Ras2, indicating that Rck1 is involved in Ras2 degradation in a proteasome-dependent manner. Furthermore, deletion of UBP3 blocked the up-regulation of FLO11, a flocculin required for pseudohyphal and invasive growth induced by Rck1 over-expression in S. cerevisiae. Taken together, these results demonstrate that Rck1 promotes S. cerevisiae pseudohyphal growth via the activation of Ubp3 phosphorylation.

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Contents

General Abstract………………………………………………..……………….1

Abstract in korean…….………….……………………………….………..……4

Contents………………..……………………………….………………………..7

List of tables…………………………………………………………...………..11

List of Figures……………………………………………………………...…...12

General Introduction………………………………………………...…………14

Objective……………………………………………………………....………..26


Part Ⅰ
Physical interaction between Sit1 and Aft1 up-regulates FOB uptake activity by inhibiting protein degradation of Sit1 in Saccharomyces cerevisiae………………………………………………………………………27
Abstract…………………………………………………………………….……28

Introduction………………………………………….……………….…………30

Materials and Methods………………………………………………….……..33
1. Strains, media, and growth conditions…………………..…………33
2. Plasmid…………………………………………………….………….33
3. Site-directed mutagenesis……………………………..……………34
4. FOB uptake Assay……………………………………….…….…....35
5. Plate assay……………………………………………….…………..36
6. Northern blot analysis…………………………………..……………36
7. Western blot analysis………………………………….…………….37
8. Microscopy…………………………………………..……………..…37
9. Sucrose gradient fractionation……………………….……………..38

Results……………………………………………………………….………….41
1. Aft1 affects the cellular localization of Sit1 and FOB uptake activity…………………………………………………..……………..41
2. Localization of Aft1 affects the FOB uptake activity of Sit1…..….46
3. Aft1 affects stability of Sit1 protein……………....…………...……51
4. Aft1 interacts with Sit1 via tyrosine 179……………………….…..54
5. Physical interaction between Aft1 and Sit1 is an important factor for FOB uptake activity and stability of Sit1…………………....….58
6. The protein stability of Sit1 via protein-protein interaction with Aft1 depends on proteasome and vacuole dependent protein degradation…………………………………………………...………61

Discussion………………………………………………………………………66


Part Ⅱ
Rck1 promotes pseudohyphal growth via the activation of Ubp3 phosphorylation in Saccharomyces cerevisiae……………………………..71

Abstract………………………………………………………………….………72

Introduction………………………………………….………………….………73

Materials and Methods………………………………………………….……..75
1. Strains, media, and growth conditions……………………………75
2. Plasmid………………………………………………………….…….75
3. Pseudohyphal growth………………………………………..………76
4. Haploid invasion assays………………...…………………..……....77
5. Plate assay………………………………………………….………..77
6. Northern blot analysis…………………………………..……………78
7. Immunoblot analysis.…………………………..…………………….78
8. Immunoprecipitation………………………………………..……..…79
9. In vivo Ubp3 phosphorylation assays……………………..……….80

Results…………………………………………………….…………………….83
1. Rck1 mRNA level is regulated by iron concentration……..….…..83
2. Rck1 regulates ubiquitination of Ras2………..………...………….86
3. Rck1 physically interacts with Ubp3 …….…………….…………..90
4. Rck1 activated phosphorylation of Ubp3………….…………...….94
5. Rck1 promotes invasive and pseudohyphal growth by activating phosphorylation of Ubp3…………...………………………………..98
6. Function of Rck1 relates with iron ………………………..………104

Discussion……………………..………………………………………………108

General Conclusion……………………………………….………………….112

Reference………………………………………………..………………….…116

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