Structural determinants specific for retromer protein sorting nexin 5 in regulating subcellular retrograde membrane trafficking

Cell culture and transfection

All cells used in the current study were cultured in a 37 ℃ incubator with 5% CO ₂. HeLa and COS-7 cells were maintained in high glucose Dulbecco's Modified Eagle Medium (DMEM, Gibco, Carlsbad, USA) supplemented with 10% fetal bovine serum (FBS, HyClone, Logan, USA) and 1% penicillin/streptomycin (Gibco). PC12 cells were maintained in DMEM supplemented with 10% defined equine serum (DES, HyClone), 5% FBS, and 1% penicillin/streptomycin. Stable lines of rat 3Flag-VMAT2 were prepared as previously described ^{[ 16]} .

Plasmid constructs and siRNA transfection were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, USA) according to the manufacturer's protocol. The transfected cells were harvested and analyzed 24 to 48 h later.

Plasmid constructions and mutagenesis

The 3Flag-TacM plasmid was constructed as previously described ^{[ 17]} . 3HA-SNX5, 3HA-SNX6, and 3HA-VMAT2 were subcloned into the pcDNA3.1-3HA vector using Kpn Ⅰ and Not Ⅰ multicloning sites. Plasmids for a series of chimeric proteins, such as 3HA-SNX6 ^5A, 3HA-SNX6 ^5B, 3HA-SNX6 ^5C, 3HA-SNX6 ^A+BAR, 3HA-SNX6 ^B+BAR, 3HA-SNX6 ^C+BAR, 3HA-SNX5 ^B+BAR, 3HA-SNX6 ^5A1, 3HA-SNX6 ^5A2, 3HA-SNX6 ^5A3, 3HA-SNX6 ^5C1, and 3HA-SNX6 ^5C2, were constructed using PCR to amplify the cDNA inserts followed by their subcloning into pcDNA3.1-3HA vector in the lab using Kpn Ⅰ and Not Ⅰ multicloning sites. The series of point mutants, such as 3HA-SNX5 (Y132D), 3HA-SNX5 (F136D), 3HA-SNX6 (A30P) ^PX, 3HA-SNX6 (S37P) ^PX, 3HA-SNX6(N62P) ^PX, 3HA-SNX6 (M143S) ^PX, 3HA-SNX6 (C149Q) ^PX, 3HA-SNX6 (R158S) ^PX, 3HA-SNX6(L161R) ^PX, and 3HA-SNX6 ^tetra-Mut, were generated by overlay site-directed mutagenesis for generating inserts followed by their subcloning into pcDNA3.1-3HA. Similarly, the constructs of His-recombinant proteins, such as 6His-SNX5 ^PX, 6His-SNX6 ^PX, 6His-SNX5-A ⁵, 6His-SNX5-B ⁵, 6His-SNX5-C ⁵, 6His-SNX6-A ⁶, 6His-SNX6-B ⁶, and 6His-SNX6-C ⁶, were subcloned into pET28a(+) vector using Nde Ⅰ and BamH Ⅰ multicloning sites. All constructions were confirmed by DNA sequencing.

Antibodies and siRNA

The primary antibodies used in the study were rabbit polyclonal anti-HA (1∶2000 dilution; Cat. #923501, Biolegend, San Diego, USA), mouse monoclonal anti-HA.11 (1∶2000 dilution; Cat. #901513, Biolegend), rabbit polyclonal anti-Flag (1∶2000 dilution; Cat. #F7425, Sigma-Aldrich, St. Louis, Missouri, USA), mouse monoclonal anti-Flag M2 (1∶2000 dilution; Cat. #F3165, Sigma-Aldrich), rabbit polyclonal anti-secretogranin Ⅱ (SgⅡ) (1∶1000 dilution for Western blotting, 1∶300 for immunofluorescence; Cat. #20357-1-AP, Proteintech, Chicago, USA), sheep polyclonal anti-TGN46 (1∶300 dilution; Cat. #AHP500, AbD Serotec, Kidlington, UK), mouse monoclonal anti-SNX5 (F-11) (1∶500 dilution; Cat. #sc-515215, Santa Cruz, Texas, USA), mouse monoclonal anti-SNX6 (D-5) (1∶500 dilution; Cat. #sc-365965, Santa Cruz), mouse monoclonal anti-β-actin (1∶5000 dilution; Cat. #66009, Proteintech), and mouse monoclonal anti-GAPDH (1∶5000 dilution; Cat. #60004, Proteintech). Secondary antibodies used were goat anti-mouse IgG (H+L) (1∶2000 dilution; Cat. #115-035-003) and goat anti-rabbit IgG (H+L) (1∶2000 dilution; Cat. #111-035-003) conjugated to HRP (Jackson, Pennsylvania, USA) as well as Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) (Cat. #A-11001), Alexa Fluor 568-conjugated goat anti-rabbit IgG (H+L) (Cat. #A-11011), and Alexa Fluor 488-conjugated donkey anti-sheep IgG (H+L) (Cat. #A-11015, Thermo Fisher Scientific, Massachusetts, USA) that were diluted in 1∶300.

The siRNA oligonucleotides and corresponding scramble siRNA were obtained from GenePharma (Shanghai, China) and resuspended in double-distilled water according to the manufacturer's instructions. Sequences used for human SNX5 siRNA interference were 5′-GCUGCUAAGGAUCUCUUAUTT-3′ and 5′-GCUUACAUAGCCUGGCUUUTT-3′. The sequences for human SNX6 were 5′-GGAACUGGCAGAGUUAGAATT-3′. The sequences used for rat Snx5 were 5′-GUGGCAGCAUUUCGAAAGATT-3′ and 5′-GCUGCAUUGAUUUAUUCAATT-3′. The sequences used for the rat Snx6 were 5′-CAGGACUCCACAGAUAUAUTT-3′ and 5′-GGCUUCAUGAUUCCUUUGUTT-3′.

Western blotting

Western blotting was performed as previously described ^{[ 18]} . Briefly, equal amounts of protein samples in sample buffer (Tris-HCl, pH 6.8, 30% glycerol, 10% SDS, 0.6 mmol/L DTT, and 0.012% bromophenol blue) were separated by electrophoresis through discontinuous 10% SDS polyacrylamide gels and transferred to a nitrocellulose membrane (Merck Millipore, Saint Charles, USA). The membrane was then blocked in Tris-buffered saline (TBS) containing 0.1% Tween-20 and 5% non-fat dry milk for 30 min at room temperature (RT) and incubated with primary antibody at 4 ℃ overnight. The next day, the membrane was washed in TBST and incubated in an appropriate secondary antibody conjugated to HRP, followed by washing in TBST and visualization by an enhanced chemiluminescence with the Tanon 5200 gel imaging system (Tanon, Shanghai, China).

Immunoprecipitation (IP)

The transfected cells were washed with ice-cold phosphate-buffered saline (PBS) once and then lysed in lysis buffer (150 mmol/L NaCl, 50 mmol/L Tris-HCl, pH 8.0, 5 mmol/L EDTA, and 0.4% NP-40) containing protease inhibitors on ice. Cell debris was centrifuged at 15000 g for 5 min, and the supernatants were collected and incubated with antibodies for 2 h at 4 ℃ followed by incubation with 30 μL pre-washed protein A/G agarose (Thermo Fisher Scientific) for an additional 2 h. After immunoprecipitation, the beads were washed in lysis buffer three times and then eluted in sample buffer before subjecting to SDS-PAGE analysis.

Recombinant protein expression and purification

The expression and purification of recombinant proteins were performed as previously described ^{[ 19]} . Briefly, His-tagged SNX5 ^PX, SNX6 ^PX, SNX5-A ⁵, SNX5-B ⁵, SNX5-C ⁵, SNX6-A ⁶, SNX6-B ⁶, and SNX6-C ⁶ were produced in Escherichia coli BL21 cells using 0.4 mmol/L isopropyl-beta-D-thiogalactopyranoside (IPTG; Biosharp, Beijing, China) for induction at 16 ℃ for overnight. The recovered bacteria were lysed with sonication in lysis buffer (1% Triton X-100 in PBS containing protease inhibitors). The fusion proteins were purified with Ni-NTA agarose (Qiagen, San Francisco, CA, USA). The recombinant proteins were detected by Coomassie brilliant blue.

His pull-down assay

For the pull-down of Flag-tagged TacM with His-fusion proteins of SNX5, SNX6, and their truncations, cells were transfected with plasmids for overexpression of Flag-TacM. Then, cells were collected and lysed using lysis buffer 24 h after transfection. The clear cell lysates prepared were incubated with equal amounts of His-fusion proteins for two hours at 4 ℃. His-fusion proteins were recovered by incubating with Ni-NTA agarose. The bound proteins on the beads were eluted using 2× sample buffer and separated by SDS-PAGE for immunoblotting using an anti-Flag antibody.

Equilibrium density gradient fractionation

Stably transformed or transiently transfected PC12 cells were harvested in buffer A (in mmol/L: 150 NaCl, 1 EGTA, 0.1 MgCl ₂, and 10 Hepes, pH 7.4) with a proteinase inhibitor. Cells were homogenized by eight passes through a ball-bearing device (clearance 12 µm). Postnuclear supernatants were then loaded onto continuous density gradients prepared with a gradient mixer using 0.65 mol/L and 1.55 mol/L sucrose in buffer B (in mmol/L: 1 EGTA, 1 MgCl ₂, and 10 Hepes, pH 7.4) and centrifuged in an SW41 rotor (Beckman Instruments, Fullerton, CA, USA) at 153900 g for 16 to 18 h at 4 ℃. Fractions (0.5 mL) were collected from top to bottom, and equal amounts of each were denatured in 6× SDS sample buffer and separated and detected by Western blotting.

Immunofluorescence and confocal microscopy

For cell staining, the process was performed as previously described ^{[ 20]} . In brief, cells were seeded on coverslips coated with poly-D-lysine (PDL, Millipore/Sigma, St. Louis, USA) and Matrigel (BD Biosciences, Franklin Lakes, USA). After being transfected by Lipofectamine 2000 for 24 to 48 h, cells were fixed with 4% PFA (Biosharp) in PBS at RT for 15 min followed by permeabilizing and blocking at RT for 15 min in a blocking solution (2% BSA, 1% fish skin gelatin, and 0.02% saponin in PBS). Cells were then incubated for two hours at RT with primary antibodies, and washed three times in the blocking solution, followed by incubation with appropriate Alexa 488 or 568-conjugated secondary antibodies for two hours. After washing again three times, coverslips were mounted on glass microscope slides using the Fluorescent Mounting Medium (Thermo Fisher Scientific).

For confocal laser microscopy, the staining was visualized with a confocal laser microscope (Carl Zeiss, LSM 710, Oberkochen, Germany), and the images were processed using the ImageJ or ZEN program.

Statistical analysis

Statistical analyses were performed using the GraphPad Prism software (version 7.0). The data were presented as mean ± standard error of the mean from at least three independent experiments with similar results. For quantitative analysis of immunoblots, the expression levels of proteins were quantified by densitometry of the bands using Image J. For the quantification of immunofluorescence images, the number of cells used for each representative experiment was indicated. One-way ANOVA was used to calculate P values for multiple group analysis. P < 0.05 was considered statistically significant.

SNX5 and SNX6 selectively regulated the trafficking of CI-MPR and VMAT2

Previously, we found that SNX5, but not SNX6, interacted with VMAT2. Although SNX5 and SNX6 share 79% of the residues in the rat protein ( Supplementary Fig. 1 , available online), they differ in the phosphoinositide binding spectrum, subcellular localization, and binding proteins. To examine the structural determinants essential for their functional differences, we first demonstrated whether SNX5 and SNX6 played distinct roles in regulating retrograde trafficking of two well-characterized cargo proteins, CI-MPR and VMAT2. Because both membrane proteins were characterized for their dependence on the C-terminus for membrane trafficking, we thus used the chimeric proteins generated in our laboratory by fusing the C-terminus of CI-MPR and VMAT2 with the Tac protein (interleukin-2 receptor α-subunit), namely Tac-MPR and TacM (Tac-VMAT2), respectively ^{[ 21– 22]} . In HeLa cells, the overexpressed Flag-tagged SNX5 and SNX6 showed similar binding affinity to the HA-tagged Tac-MPR from the co-immunoprecipitation (co-IP) experiment ( Fig. 1A ). However, SNX5, but not SNX6, showed a significantly higher binding affinity to VMAT2 ( Supplementary Fig. 2A , available online), while immunofluorescent staining showed that SNX5 and SNX6 partially colocalized with VMAT2, indicating the transient interaction ( Supplementary Fig. 2B ). Furthermore, similar results were also achieved by using the chimeric protein TacM, which showed a high affinity in binding to SNX5, but not SNX6 ( Fig. 1B and 1C ). To examine the physiological significance of this interaction, we used siRNA-mediated knockdown for SNX5 or SNX6 in HeLa cells ( Supplementary Fig. 3 , available online) and the results showed that the depleted expression of SNX5 and SNX6 altered the subcellular localization of CI-MPR ( Fig. 1D ). On the contrary, only the reduced expression of SNX5, but not SNX6, altered subcellular colocalization of VMAT2 with trans-Golgi network protein 46 (TGN46; Fig. 1E ). These results suggested that SNX5 and SNX6 were involved in different sorting pathways.

The N-terminal PX domain of SNX5 or SNX6 was thought to interact with phosphoinositide; but more recently, it has been found to interact directly with proteins ( Fig. 2A ) ^{[ 23– 24]} . Interestingly, we have previously shown that the N-terminal PX domain of SNX5 is responsible for its interaction with VMAT2 ^{[ 25]} . Because the PX domain of SNX5 and SNX6 shares a high similarity ( Supplementary Fig. 1 ), we assessed their interactions with TacM using His-tagged recombinant PX domain of SNX5 and SNX6. As shown in Fig. 2B , only His-SNX5 ^PX, but not SNX6 ^PX, pulled down TacM from the cell extracts. We also found that the overexpressed PX domain of SNX5, but not SNX6, functionally disrupted the TGN46 colocalization with VMAT2 ( Fig. 2C ), strongly indicating that the PX domain of SNX5 and SNX6 may contain structural determinants for their distinct affinity in binding to VMAT2 and regulating its membrane trafficking.

The PX domains of SNX5 and SNX6 contain distinct residues for protein binding

To determine the key residues of the PX domain of SNX5 or SNX6 essential for recognizing cargos, we used the chimeric mutagenesis approach. Based on the structural features of the PX domain from previous studies and the sequence differences of the PX domain between SNX5 and SNX6 ( Supplementary Fig. 1 ) ^{[ 10, 26– 27]} , we divided their PX domains into three fragments: head group A ^5/6 (1–90), two-helix loop B ^5/6 (91–140), and helix linker C ^5/6 (141–181). We genetically generated the chimeric proteins by swapping these fragments between the two protein PX domains accordingly ( Fig. 3A ). Using a co-IP assay to examine the binding affinity of these constructs with TacM, we noticed that replacing A ⁶ or C ⁶ fragments of SNX6 with those of SNX5, respectively (SNX6 ^5A and SNX6 ^5c), significantly restored the binding affinity of the chimeric protein SNX6 with TacM, comparable with that of wild type SNX5 ( Fig. 3A ). Notably, the helix loop (SNX6 ^5B) chimeric protein failed to restore the binding of SNX6 and TacM. These results suggest one of the following two possibilities: the fragments A and C of SNX5, but not SNX6, might contain residues essential for their binding to VMAT2, or the fragment B of both SNX5 and SNX6 might include the binding residue, while the fragment A and C of SNX6 contained inhibitory residues to inhibit the interaction between SNX6 and VMAT2. It is worth mentioning that 3Flag-TacM from the input sample was shown in two bands ( Fig. 3A ), but not from the co-IP samples, which may be a result of the binding preference of protein A/G agarose for the lower molecular weight form of the protein. The nature of the two bands of 3Flag-TacM remains unclear, although the Tac protein (IL2RA) has been shown to undergo posttranslational modification by glycosylation.

The analysis of the special motif within SNX5 required for its interaction with VMAT2

To examine which mechanism is involved in the binding of PX domain to VMAT2, we overexpressed chimeric protein containing each fragment and BAR domain from SNX6 PX domain separately, and found that only the fragment B ⁶ might be able to interact with TacM by co-IP ( Fig. 3B ). However, the expression levels of SNX6 ^A+BAR and SNX6 ^C+BAR were very low, which might be due to their instability or cytotoxicity in cells ^{[ 28]} . To further confirm their binding specificity, we used an in vitro pull-down assay by using these individual recombinant protein fragments. As shown in Fig. 3C , only fragment B of both SNX5 and SNX6 specifically pulled down TacM. In particular, we tested two point mutations at the Y132D and F136D sites in the B ⁵ fragment, which were reported to be required for the specific binding between SNX5 and CI-MPR ^{[ 23]} . As shown in Fig. 3D , these mutants led to the deprivation of their interaction with TacM, further validating the specificity and importance of the B fragment for the recognition and binding of cargo proteins.

Taken together, these results strongly supported our second hypothesis that the B fragment of both SNX5 and SNX6 had a special binding affinity for VMAT2, which was inhibited, in SNX6, by the A and C fragments. Thus, these two fragments could contain specific inhibitory residues in SNX6 to block the interaction of its PX domain with VMAT2.

Identification of key residues in SNX6 required to inhibit its interaction with VMAT2

To screen for the region and residues in the A and C fragments of the SNX6 PX domain that are responsible for inhibition, we further divided these fragments into five subregions to generate chimeric proteins. The A fragment was divided into three subregions as A1, A2, and A3 with the corresponding amino acid sequences as 1–35, 36–57, and 58–90, respectively, and the C fragment into two subregions as C1 and C2 with the corresponding amino acid sequences as 141–160 and 161–181 ( Fig. 4A ), respectively. The corresponding subregions of SNX6 were replaced by those of SNX5 for a set of chimeric proteins to examine their binding affinity to TacM using the co-IP assay. As shown in Fig. 4B , compared with SNX6 ^WT, chimeric proteins tagged with HA, SNX6 ^5A2, SNX6 ^5A3, and SNX6 ^5C1, showed partial binding to TacM, suggesting that these three subregions in SNX5, altered in SNX6, contained the key motifs required for specific binding to VMAT2. Therefore, based on the differences in the amino acid sequence of these three subregions between SNX5 and SNX6 and the structural significance of prolines in the PX domain, we focused on mutagenizing amino acids in SNX6 corresponding to those in SNX5 at the A30, S37, N62, M143, C149, R158, and L161 sites ( Fig. 4C ). As shown in Fig. 4D and 4E , mutants with point mutations of S37P, N62P, M143S, R158S, and L161R of SNX6 clearly showed a restoration of their binding to TacM, compared with that of SNX6 ^WT, suggesting that these four residues in SNX5 played a critical role in the specific interaction with VMAT2. On the contrary, the altered residue sequence for these four amino acids in SNX6 may be responsible for the altered structure of these subregions required for the interaction. Furthermore, these results support the idea that SNX5 and SNX6 may play different regulatory functions in retrograde membrane trafficking of VMAT2.

To test whether these key residues are functionally critical for SNX5 but not for SNX6 in regulating the membrane trafficking of VMAT2, we first generated a tetramutant SNX6 construct (SNX6 ^tetra-Mut), in which the following residues, S37, N62, M143, and R158, were mutated to S37P, N62P, M143S, and R158S in the corresponding amino acid for SNX5 ( Fig. 5A ). As shown in Fig. 5B , the SNX6 tetramutant showed a strong interaction with TacM, compared with wild-type SNX6. Furthermore, we made several silent nucleotide mutations to allow this construct to be resistant to specific SNX5 siRNA ( Fig. 5C ). Consistent with the previous results, the loss of SNX5 expression induced by siRNA-mediated knockdown altered the subcellular localization of VMAT2 away from TGN. However, overexpressed SNX6 ^tetra-Mut completely rescued the mistargeting of VMAT2, presumably by restoring the retrograde trafficking of the transporter ( Fig. 5D ).

Key residues in SNX5-PX essential for regulating the large dense core vesicles (LDCVs) targeting of VMAT2 in PC12 cells

We then examined the functional relevance of the key residues of SNX6 (Ser ³⁷, Asp ⁶², Met ¹⁴³ and Arg ¹⁵⁸), identified in the mutagenesis study in VMAT2 targeting to LDCVs using PC12 cells. PC12 stable transformants expressing the Flag-tagged VMAT2 were first studied, upon siRNA-mediated knockdown of endogenous Snx5, and it showed a disrupted colocalization of VMAT2 with SgⅡ, the marker for LDCVs, at the tips of neural processes ( Fig. 6A and Supplementary Fig. 4 [available online]). However, the loss of Snx6 expression had no effect on the LDCV targeting of VMAT2. Consistently with its biochemical results, overexpressed SNX6 ^tetra-Mut clearly restored vesicular targeting of VMAT2 to LDCVs, which was altered in control cells after Snx5 knockdown ( Fig. 6A and Supplementary Fig. 4 ), strongly supporting the notion that these key residues in SNX5 were important for structural-dependent protein interaction and function. Furthermore, the vesicular localization of VMAT2 to LDCVs, required for its transport activity, was confirmed by the pharmacological analysis. As shown in Fig. 6B , the depleted expression of endogenous SNX5, but not SNX6, significantly decreased the depolarization-dependent release of preloaded ³H-norepinephrine ( ³H-NE) from PC12. Since NE is one of the monoamine transmitter substrates for VMAT2 and normally is packaged inside of both synaptic vesicles and LDCVs in neurons, mostly in LDCVs in PC12 cells, this result indicated that the altered VMAT2 membrane targeted to non-secretory subcellular membrane compartments. Consistently, SNX6 ^tetra-Mut restored the stimulated release of ³H-NE in Snx5 knockdown cells, suggesting that the SNX5-like SNX6 ^tetra-Mut functionally restored the LDCV targeting of VMAT2.

The effect of SNX6 ^tetra-Mut on the subcellular membrane trafficking of VMAT2 was further examined biochemically by using the density gradient fractionation of PC12 transformants. As shown in Fig. 6C , HA-VMAT2 was identified in fractions containing the LDCV marker SgⅡ in heavy fractions in control cells, but it showed an altered distribution to light fractions after Snx5 knockdown, suggesting the comigration of two proteins in LDCV. Similarly, overexpressed SNX6 ^tetra-Mut restored the LDCV targeting of VMAT2 due to the loss of SNX5. In conclusion, both immunofluorescent staining and gradient fractionation analysis provided biochemical and cellular evidence that residues, such as Pro ³⁶, Pro ⁶¹, Ser ¹⁴² and Ser ¹⁵⁷ in the PX domain of SNX5, were structural determinants not only for their interactions with VMAT2 but also for the functional regulation of vesicular targeting via retrograde trafficking of the transport protein to LDCVs for the secretory process in both endocrine glands and monoamine neurons. Without these residues, SNX6 no longer occupied the structure of PX domain essential for its interaction with VMAT2 and the functional regulatory role in the targeting of VMAT2 to LDCVs.