Journal: bioRxiv

Article Title: Endocytosis of a zinc transceptor ZIP4 is mediated by AP2 through an atypical dileucine motif

doi: 10.1101/2025.05.03.652025

Figure Lengend Snippet: Effects of the LQL mutations on human ZIP4 endocytosis in HEK293T cells. ( A ) Topology of hZIP4-HA in the plasma membrane. The LQL segment is located in the IL2 loop. The ECD is shown as a green oval. The HA tag (orange rectangle) is exposed to the extracellular space. ( B ) Effects of substitutions of the residues in the LQL segment on human ZIP4-HA endocytosis. E458 is a generally conserved residue in ZIP4 and thus was tested along with the LQL sequence. The internalized mouse anti-HA antibodies were detected by Western blot using an HRP-conjugated anti-mouse antibody. The total expression levels of ZIP4 variants were detected by an anti-HA antibody. Western blot of anti-β actin was used as loading control. ( C ) Comparison of anti-HA antibody uptake between wild-type ZIP4-HA and its variants using a flow cytometry-based assay. ΔMFI was calculated by subtracting the MFI of the cells incubated with the antibody at 4°C from the MFI of the cells incubated with the antibody at 37°C. The values of ΔMFI were then normalized by setting the value for wild-type ZIP4-HA as 100%. Each column chart shows the data in one of three independent experiments with three biological replicates tested for each condition. The representative histograms on the right show the profiles of 4°C (shaded) and 37°C (open). Statistical analyses were performed using two-tailed Student’s t -test. The P values are 0.0011 (L452A) 0.00099 (L452N), 0.015 (L454I), 0.0012 (L454A), and 0.00033 (L454N), respectively. *: P <0.05; **: P <0.01; ***: P <0.001.

Article Snippet: The pCMV3-based vectors for the expression of N-terminal FLAG-tagged mouse ZIP4 (GenBank ID: 72027, Cat# MG53693 - NF) and the untagged human AP2S1 (σ2 subunit of AP2, GenBank ID: 1175, Cat# HG12478 -UT) in mammalian cells were purchased from Sino Biological.

Techniques: Clinical Proteomics, Membrane, Residue, Sequencing, Western Blot, Expressing, Control, Comparison, Flow Cytometry, Incubation, Two Tailed Test

Journal: bioRxiv

Article Title: Endocytosis of a zinc transceptor ZIP4 is mediated by AP2 through an atypical dileucine motif

doi: 10.1101/2025.05.03.652025

Figure Lengend Snippet: Endocytosis of endogenously expressed ZIP4 in HepG2 cells. ( A ) Internalization of the 1G3B2 anti-ZIP4 mAb by HepG2 cells. Live HepG2 cells were incubated with 1G3B2 mAb at 4°C and 37°C, respectively. After wash, fixation and permeabilization, cells were stained with an Alexa Fluor-568 conjugated anti-mouse antibody. Immunofluorescence images were taken using a confocal microscopy. Representative cells from multiple images were shown. Scale bars indicate 10 μm. ( B ) Analysis of 1G3B2 mAb uptake by flow cytometry. The target proteins were knocked down by siRNA. ΔMFI was calculated by subtracting the MFI of the cells incubated with the antibody at 4°C from the MFI of the cells incubated with the antibody at 37°C. The values of ΔMFI were then normalized by setting the value of the control group as 100%. The column chart shows the data in one of two independent experiments with three biological replicates tested for each condition. The representative results are shown in histogram chart on the right. Statistical analyses were conducted using two-tailed Student’s t -test. The P values are 0.0068 (ZIP4), 0.0013 (CHC), 0.0047 (AP2-α), and 0.0017 (AP2-σ2), respectively. **: P <0.01. ( C ) Knockdown of the target proteins by control siRNA or specific siRNA. The expression levels were analyzed in Western blot using the corresponding antibodies.

Article Snippet: The pCMV3-based vectors for the expression of N-terminal FLAG-tagged mouse ZIP4 (GenBank ID: 72027, Cat# MG53693 - NF) and the untagged human AP2S1 (σ2 subunit of AP2, GenBank ID: 1175, Cat# HG12478 -UT) in mammalian cells were purchased from Sino Biological.

Techniques: Incubation, Staining, Immunofluorescence, Confocal Microscopy, Flow Cytometry, Control, Two Tailed Test, Knockdown, Expressing, Western Blot

Journal: bioRxiv

Article Title: Endocytosis of a zinc transceptor ZIP4 is mediated by AP2 through an atypical dileucine motif

doi: 10.1101/2025.05.03.652025

Figure Lengend Snippet: Structural model of the AP2-IL2 complex generated by AlphaFold 3. ( A ) Overall structure ( left ) and zoomed-in view ( right ). AP2 is shown in surface mode and the ZIP4-IL2 peptide (438-494, pink) is in cartoon mode ( left ). 14 of 35 predicted models showed a consistent binding of the LxL motif to a hydrophobic pocket in the σ2 subunit ( right ). L452 and L454 are depicted in stick mode. ( B ) Comparison of the binding of the LxL motif ( left ) and the canonical dileucine motif in CD4 ( middle , PDB: 2JKR). The superimposed structures are shown in the right panel. The residues mutated in this work (V88, L103, N9, R15, and E100 in the σ2 subunit) are underlined.

Article Snippet: The pCMV3-based vectors for the expression of N-terminal FLAG-tagged mouse ZIP4 (GenBank ID: 72027, Cat# MG53693 - NF) and the untagged human AP2S1 (σ2 subunit of AP2, GenBank ID: 1175, Cat# HG12478 -UT) in mammalian cells were purchased from Sino Biological.

Techniques: Generated, Binding Assay, Comparison

Journal: bioRxiv

Article Title: Endocytosis of a zinc transceptor ZIP4 is mediated by AP2 through an atypical dileucine motif

doi: 10.1101/2025.05.03.652025

Figure Lengend Snippet: Identification of the key residues in the AP2-IL2 interaction. ( A ) The importance of the hydrophobic pocket in the σ2 subunit. The σ2 subunit in HepG2 cells was knocked down by siRNA and rescued by overexpressing an siRNA-resistant σ2 subunit or its variants with hydrophobic residues (V88 and L103) substituted with polar/charged amino acids. ΔMFI was calculated by subtracting the MFI of the cells incubated with the antibody at 4°C from the MFI of the cells incubated with the antibody at 37°C. The values of ΔMFI were then normalized by setting the value of the control group as 100%. The representative results are shown in histogram chart ( middle ). Statistical analyses were conducted using two-tailed Student’s t -test. The P values from left to right are 0.00020, 0.00056, 0.0020, 0.031, and 0.0011, respectively. *: P <0.05; **: P <0.01; ***: P <0.001. The expression levels of the wild-type σ2 subunit or its variants were examined by Western blot, and β-actin was used as loading control. ( B ) Examination of the involvement of the electrostatic patch (composed of N9, R15, and E100) in the σ2 subunit. The knockdown and rescue experiments and Western blot were conducted in the same as in (A). The P values from left to right are 0.00043, 0.00027, and 0.0035, respectively. ( C ) Endocytosis of wild-type ZIP4-HA and its variants with changed distance between the two leucine residues in the LxL motif. The constructs were transiently expressed in HEK293T cells and the anti-HA antibody uptake assay was conducted and detected as described in . For A, B , and C , the column chart ( left ) shows the data in one of two independent experiments with three biological replicates tested for each condition.

Article Snippet: The pCMV3-based vectors for the expression of N-terminal FLAG-tagged mouse ZIP4 (GenBank ID: 72027, Cat# MG53693 - NF) and the untagged human AP2S1 (σ2 subunit of AP2, GenBank ID: 1175, Cat# HG12478 -UT) in mammalian cells were purchased from Sino Biological.

Techniques: Incubation, Control, Two Tailed Test, Expressing, Western Blot, Knockdown, Construct

Journal: bioRxiv

Article Title: Endocytosis of a zinc transceptor ZIP4 is mediated by AP2 through an atypical dileucine motif

doi: 10.1101/2025.05.03.652025

Figure Lengend Snippet: Cysteine accessibility assay of mouse ZIP4 expressed in HEK293T cells. ( A ) Topology of the cysteine-free TMD of mouse ZIP4 (CysFree-TMD). The amino acid sequence of the IL2 loop is bolded and the residues substituted with cysteines are highlighted in red. The LxL motif is shown in green. Note that a cysteine residue is present at the position of 454 in wild-type ZIP4. ( B ) Cartoon illustration of the experimental procedure. ( C ) Cysteine accessibility assays of eight single cysteine variants. For each variant, an NEM titration was conducted and the red dashed line indicates the concentration of NEM above which the cysteine of interest is 100% labelled by NEM. Cells in control group (Con) were not treated with NEM or mPEG5k. An anti-FLAG antibody was used in Western blot experiments. The shown data are the results of one of two independent experiments with similar results.

Article Snippet: The pCMV3-based vectors for the expression of N-terminal FLAG-tagged mouse ZIP4 (GenBank ID: 72027, Cat# MG53693 - NF) and the untagged human AP2S1 (σ2 subunit of AP2, GenBank ID: 1175, Cat# HG12478 -UT) in mammalian cells were purchased from Sino Biological.

Techniques: Sequencing, Residue, Variant Assay, Titration, Concentration Assay, Control, Western Blot

Journal: bioRxiv

Article Title: Endocytosis of a zinc transceptor ZIP4 is mediated by AP2 through an atypical dileucine motif

doi: 10.1101/2025.05.03.652025

Figure Lengend Snippet: Proposed mechanism of zinc-dependent ZIP4 endocytosis. Cellular zinc availability regulates the conformational states of the IL2 loop, where the LxL motif is located. Under zinc depletion conditions, the LxL motif is less accessible (right); with increased zinc levels, the LxL motif becomes more exposed (left), allowing it to be recruited by AP2 (PDB: 2XA7, orientated in the membrane-bound state) through a direct binding to the hydrophobic pocket in the σ2 subunit (indicated by arrow and zoomed in the inset), where canonical dileucine motifs of cargo proteins bind. For clarity, the ECD of ZIP4 is trimmed. The transport domain of ZIP4 is depicted as a two-domain protein with the scaffold and transport domains shown in green and blue, respectively. Alternating access is achieved by vertical sliding of the transport domain against the static scaffold domain, which is proposed to trigger conformational changes of the IL2 loop. Zinc ions are depicted as grey spheres.

Article Snippet: The pCMV3-based vectors for the expression of N-terminal FLAG-tagged mouse ZIP4 (GenBank ID: 72027, Cat# MG53693 - NF) and the untagged human AP2S1 (σ2 subunit of AP2, GenBank ID: 1175, Cat# HG12478 -UT) in mammalian cells were purchased from Sino Biological.

Techniques: Membrane, Binding Assay