Physical interaction between Sit1 and Aft1 upregulates FOB uptake activity by inhibiting protein degradation of Sit1 in Saccharomyces cerevisiae.
Previously, we reported that Aft1 regulates Sit1 by modulating the ubiquitination of Sit1 in Saccharomyces cerevisiae. Here, we report the function of the physical interaction between Sit1 and Aft1 in ferrioxamine B (FOB) uptake. The interaction between Sit1 and Aft1 induced protein localization of Sit1 to the plasma membrane, and more Sit1 was detected in the plasma membrane when Sit1 and Aft1 were coexpressed compared with Sit1 expression alone. The MSN5-deletion mutant, which failed to translocate Aft1 to the cytosolic compartment, showed lower FOB uptake activity than the wild type. However, higher free iron uptake activity was detected in the MSN5-deletion mutant. Furthermore, the strain transformed with AFT1-1(up) plasmid, which failed to regulate Aft1 via iron concentration and accumulated Aft1 in the nucleus, showed lower FOB uptake activity. The Aft1 Y179F mutant, which contained a tyrosine residue that was changed to phenylalanine, failed to interact physically with Sit1 and showed more degradation of the Sit1 and, ultimately, lower FOB uptake activity. Additionally, we found that MG132 and PMSF, which are inhibitors of proteasomes and serine proteases, respectively, increased the Sit1 protein level. Taken together, these results suggest that the protein-protein interaction between Sit1 and Aft1 is an important factor in the FOB uptake activity of Sit1.
School of Life Sciences and Biotechnology, Korea University, Anam-dong, Sungbuk-gu, Seoul 136-701, Republic of Korea.
Chang-Min
CM
Kang
School of Life Sciences and Biotechnology, Korea University, Anam-dong, Sungbuk-gu, Seoul 136-701, Republic of Korea.
Suzie
S
Kang
School of Life Sciences and Biotechnology, Korea University, Anam-dong, Sungbuk-gu, Seoul 136-701, Republic of Korea.
Yong-Sung
YS
Park
School of Life Sciences and Biotechnology, Korea University, Anam-dong, Sungbuk-gu, Seoul 136-701, Republic of Korea cheolwony@korea.ac.kr.
Cheol-Won
CW
Yun
D002462:Cell Membrane / Q000378:metabolism; D003676:Deferoxamine / Q000378:metabolism*; D005290:Ferric Compounds / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D059748:Proteolysis; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*
C090297:AFT1 protein, S cerevisiae; D005290:Ferric Compounds; D026901:Membrane Transport Proteins; C118510:SIT1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C002577:ferrioxamine B; D003676:Deferoxamine
Aft1; S. cerevisiae; Sit1; siderophore