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First published online 10 June 2008
doi: 10.1242/jcs.019745

Journal of Cell Science 121, 2217-2223 (2008)
Published by The Company of Biologists 2008
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Interaction of Mint3 with Furin regulates the localization of Furin in the trans-Golgi network

Jinbo Han1,2,*, Yiguo Wang1,*, Suming Wang3,* and Chengwu Chi1,2,{ddagger}

1 Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, People's Republic of China
2 Institute of Protein Research, Tongji University, Shanghai 200092, People's Republic of China
3 School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, People's Republic of China


Figure 1
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  		Fig. 1. Mint3 interacts with Furin in HeLa cells. The association of Mint3 with Furin in HeLa cell lysates is shown, as detected by co-immunoprecipitation (A) and reverse co-immunoprecipitation (B) assays. TCL (total cell lysate) is shown as 1% of input.

 

Figure 2
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  		Fig. 2. Mint3 is required for the localization of Furin to the TGN. (A) Knockdown of endogenous Mint3. HeLa cells were transiently transfected with either empty vector (control), non-target control siRNA (NT) or human Mint3 shRNA targeting plasmids (RNAi-1 and RNAi-2). Equal amounts of total protein were analyzed by immunoblot as shown by detected levels of β-actin. The quantification of the immunoblots is shown in the graph. Each value represents the mean ± s.e.m. of triplicate experiments. Asterisks denote significant differences from control cells (P<0.05), as determined by Student's t-test. (B) Loss of Mint3 significantly disrupts the localization of Furin to the TGN. Arrowheads indicate cells in which Mint3 expression was suppressed by RNAi. Bar graphs shows the proportion of cells with Mint3 knockdown (out of 100 cells counted on each of two different slides for each condition). Asterisks denote significant differences from control cells (P<0.05), as determined by Student's t-test. (C) Subcellular localization of Furin in control and Mint3-knockdown cells. HeLa cells transiently transfected with either empty vector (control) or human Mint3 shRNA plasmid (RNAi-2) were fixed and immunostained with antibodies to Furin and either TGN46, KDEL or EEA1 as indicated. The enlarged regions of the boxed areas are shown to the right. Scale bars: 5 µm.

 

Figure 3
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  		Fig. 3. The PTB domain of Mint3 is functionally required for localization of Furin in the TGN. (A) Schematic representation of Mint3 and its various deletion mutants. The binding abilities of His-tagged fusion proteins to Furin are summarized to the right based on the pull-down assays shown in B. (B) Mint3 interacts with Furin through its PTB domain. His-tagged fusion proteins were immobilized on TALON metal affinity beads and incubated with HeLa cell lysates. The upper panel shows a specific immunoblot for Furin from the pull-down lysates, and the lower panel represents the Coomassie staining of His-tagged fusion proteins in the pull-down assays. TCL (total cell lysate) is shown as 1% of input. (C) The PTB domain of Mint3 is necessary for the localization of Furin at the TGN. HeLa cells were co-transfected with pCMV-Furin, human Mint3 shRNA plasmids and with the GFP-tagged mouse Mint3 constructs indicated in the left panel. Cells were fixed and immunostained with antibodies to Furin and Mint3 as indicated. Scale bars: 5 µm.

 

Figure 4
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  		Fig. 4. Binding of the acidic peptide region of Furin by Mint3. (A) Schematic diagrams of Furin and its various deletion mutants. The binding abilities of GST-tagged fusion proteins to Mint3 are summarized to the right. ND, not determined. (B) The association of the cytoplasmic domain of Furin with Mint3. GST fusion proteins of the catalytic domain (GST-CA), the P-domain (GST-P) and the cytoplasmic domain (GST-CP) of Furin were immobilized on glutathione-Sepharose beads and incubated with lysates from HeLa cells. Bound proteins were eluted, separated by SDS-PAGE and visualized as indicated. TCL (total cell lysate) is shown as 5% of input. (C) Schematic representation of GST fusion proteins with various mutations of the Furin cytoplasmic domain. Deleted regions are marked by a dotted line and amino acid substitutions are labeled in red. Binding motifs are bold and underlined. The binding abilities of GST-tagged fusion proteins to Mint3 are summarized to the right. (D) The mutants with acidic peptide deletion ({Delta}769-780) fail to bind Mint3 efficiently, and the mutants with LI/AN Y/A substitutions decrease the binding affinity between Furin and Mint3.

 

Figure 5
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  		Fig. 5. A model of the regulation of Furin TGN localization by Mint3. Binding of the hydrophobic motif to the adaptor protein AP-1 is required for the budding of Furin from the TGN to endosomes, whereas binding of CK2-phosphorylated Furin acidic cluster to the sorting protein PACS1 transports Furin back from endosomes to the TGN (Thomas, 2002Go). In the TGN, Mint3 mainly binds to the acidic cluster of Furin and retains most of the Furin molecule in this compartment. However, binding of the YKGL and LI motifs of Furin with Mint3 might compete its binding with AP-1 and hence reduce the budding of Furin from the TGN. CA, catalytic domain; CP, cytoplasmic domain; P, P domain; TM, transmembrane domain.

 

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