Identification of Cul3-KLHL20 as a SERINC5 ubiquitin E3 ligase via mass spectrometry. To understand how SERINC5 is polyubiquitinated, we interrogated the presence of E3 ubiquitin ligases in SERINC5 protein complexes by mass spectrometry. FLAG-tagged SERINC5 was purified from HEK293T cells by an anti-FLAG affinity column and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS), as we reported18. Four independent experiments were conducted from which a total of 25 ubiquitin E3 ligase-associated proteins were identified (Fig. 1A). Cul1, Cul3, and Cul4B were found in this list. Because Cul4B is expressed in the nucleus and responsible for cell cycle regulation, only Cul1 and Cul3 were selected for our study.
We reported that ectopic ubiquitin (Ub) decreases SERINC5 expression at steady state14, 15, 16. Accordingly, we expressed SERINC5 with ubiquitin in HEK293T cells in the presence of Cul1 and/or its adaptor protein Skp1, or Cul3 and/or its adaptor protein KLHL20. The SERINC5 expression was then analyzed by western blotting (WB) (Fig. 1B). A SERINC5 lysine-free mutant with all 19 lysine residues mutated to arginine (1-19K/R), was included as a control. Ubiquitin decreased SERINC5 expression as expected (Fig. 1B, lanes 1, 2, 6, 7). Although this decrease was not affected by single expression of Cul1, Skp1, Cul3, or KLHL-20 and co-expression of Cul1 with Skp1 (lanes 3–5, 7–8), it was notably enhanced by co-expression of Cul3 with KLHL20 (lane 10). Neither ubiquitin nor Cul3/KLHL20 decreased the 1-19K/R mutant expression (lanes 11–15). In addition, KLHL20 decreased the amount of detectable ubiquitin (lanes 8, 10, 13, 15), indicating that Cul3-KLHL20 deceases the cellular ubiquitin pool. These results suggested that SERINC5 was likely targeted by the CRL3KLHL20 ubiquitin E3 ligase.
Next, we interrogated the mechanism of how SERINC5 interacts with Cul3-KLHL20. We tried to knock out Cul3 and KLHL20 in HEK293T cells after expressing Cas9 with their specific guide (g) RNAs. Although these gRNAs effectively knocked out Cul3 or KLHL20 (Fig. 1C), we could not obtain stably knockout clones due to their necessity to cell survival. Thus, we used the same sets of Cas9 and gRNAs to transiently knock out their expression and studied their interaction with SERINC5.
To confirm that the SERINC5 interacts with Cul3/KLHL20, they were expressed in HEK293T cells and SERINC5 was subjected to immunoprecipitation (IP) (Fig. 1D). SERINC5 pulled down KLHL20 and Cul3, but not the GFP control (Fig. 1D, IP, lanes 2, 3, 5), indicating that SERINC5 interacts with Cul3-KLHL20. To test whether these interactions were dependent on each other, KLHL20 and Cul3 were knocked down by CRISPR/Cas9. The SERINC5-KLHL20 interaction was not affected by Cul3-knockdonw (KD), but the SERINC5-Cul3 interaction was disrupted by KLHL2-KD (IP, lanes 4, 6). These results demonstrate that SERINC5 interacts with Cul3-KLHL20 via KLHL20.
We then tested how SERINC5 interacts with KLHL20. KLHL20 has 609 amino acids that comprise three domains including Kelch-repeat (residues 1-316), BTB and C-terminal Kelch (BACK), and Bric-a-brac/tramtrack/broad complex (BTB) (residues 301–609). In addition, six residues at positions 109, 111, 113, 146, 148, and 150 are required for its binding to Cul323. Two KLHL20 mutants, KLHL20∆K that does not have the Kelch-repeat domain, and KLHL20m6 that has thosee six residues replaced with alanine, were tested for their interaction with SERINC5 by IP (Fig. 1E). Both the wild-type (WT) KLHL20 and its m6 mutant interacted with SERINC5 (Fig. 1E, IP, lanes 2, 4), whereas the ∆K mutant did not (IP, lane 3). These results demonstrate that SERINC5 binds to KLHL20 via the Kelch-repeat domain.
Next, we investigate the role of Cul3-KLHL20 in SERINC5 polyubiquitination. SERINC5 was expressed with ubiquitin in HEK293T cells in the presence of KLHL20/Cul3 expression or their knockdown by CRISPR/Cas9. Ectopic ubiquitin was subjected to immunoprecipitation and levels of SERINC5 polyubiquitination were analyzed by WB (Fig. 1F). To avoid SERINC5 degradation by ubiquitin, a ubiquitin mutant that had its K48 and K63 mutated to arginine (UbK48/63R) was used. Polyubiquitinated SERINC5 proteins were detected at over 90 kDa, that were increased by KLHL20 and/or Cul3 (Fig. 1F, IP, lanes 2–4). In addition, both KLHL20-KD and Cul3-KD notably reduced the SERINC5 polyubiquitination (IP, lanes 2, 6, 9). Importantly, this reduction by knockdown was rescued by ectopic KLHL20 or Cul3 expression (IP, lanes 7, 10).
To further explore this mechanism, the SERINC5 polyubiquitination was re-analyzed in the presence of ectopic ubiquitin, KLHL20 proteins, and the Cul3 E2-recruiting protein Rbx1 (Fig. 1G). When SERINC5 expression in cells was compared, its decrease by ubiquitin was enhanced by KLHL20 and Rbx1 (Fig. 1G, Input, lanes 3, 6), but not by the KLHL20∆K and KLHL20m6 mutants (Input, lanes 4, 5). Consistently, when SERINC5 polyubiquitination was compared, it was increased by KLHL20 and Rbx1 (IP, lanes 3, 6), but was strongly decreased by these two KLHL20 mutants (IP, lanes 4, 5). These results demonstrate that like KLHL20, Rbx1 is also required for SERINC5 polyubiquitination. In addition, the SERINC5 polyubiquitination depends on the Cul3-KLHL20 interaction and the Kelch-repeat domain. Furthermore, it was noticeable that the ∆K mutant was not detected from the pulldown sample (IP, lane 4), which further confirms that SERINC5 interacts with KLHL20 via the Kelch-repeat domain. Altogether, these results demonstrate that CRL3KLHL20 is a SERINC5-associated ubiquitin ligase that is responsible for SERINC5 polyubiquitination.
Identification of lysine 130 (K130) as a critical polyubiquitination site on SERINC5. The 423 amino acids of human SERINC5 comprise 5 extracellular loops (ECLs), 10 transmembrane domains (TMDs), and 4 intracellular loops (ICLs) (Fig. 2A). They also include 19 lysine residues (K1 to K19) that are found spread throughout these regions. To test if these lysine residues are targeted for polyubiquitination, we generated SERINC5 mutants in which these lysine residues were replaced with arginine and determined how levels of SERINC5 polyubiquitination are affected.
Initially, we tested how ubiquitin affected expression and polyubiquitination of mutants 1-7K/R, 1-11K/R, 1-14K/R, 15-19K/R, and 1-19K/R, which have K1-K7, K1-K11, K1-K14, K15-K19, or K1-K19 replaced with arginine. These SERINC5 lysine mutants were expressed with ectopic ubiquitin and immunoprecipitated by anti-FLAG that targets SERINC5, and their polyubiquitination was detected by anti-HA that detects ectopic ubiquitin (Fig. 2B). Ubiquitin reduced expression of SERINC5 WT, 1-7K/R, and 15-19K/R in cells, and consistently, high levels of polyubiquitinated SERINC5 products were detected from these proteins (Fig. 2B, lanes 1–4, 9–10). On the contrary, ubiquitin did not affect expression of 1-11K/R, 1-14K/R, and 1-19K/R in cells, and consistently, their polyubiquitinated products were detected at much reduced levels (lanes 5–8, 11–12). These results suggest that there is a specific SERINC5 lysine residue(s) responsible for its polyubiquitination.
To further narrow down the specific site of polyubiquitination, we mutated K8 and K9, which are located at residue 130 or 179, to arginine, by generating mutants K130R and K179R. When these two mutants were expressed with ubiquitin, expression of K179R (lanes 17–18), but not K130R (lanes 15–16), was decreased in cells. In addition, K179R had a similar level of polyubiquitination as WT SERINC5 (lanes 13–14, 17–18), whereas K130R was poorly polyubiquitinated (lanes 15–16). These results demonstrate that K130 is the critical site for SERINC5 polyubiquitination.
To understand how K130 is polyubiquitinated, we generated a SERINC5 mutant, designated 130K, in which all the lysine residues, except K130, were replaced with arginine. In addition, we generated seven ubiquitin mutants, UbK6R, UbK11R, UbK27R, UbK29R, UbK33R, UbK48R, and UbK63R, where each of the seven lysine residues in ubiquitin were individually mutated to arginine. We also generated a ubiquitin mutant, UbKO, in which all seven lysine residues were mutated to arginine. When the SERINC5-130K mutant was expressed with either WT ubiquitin (UbWT) or each of the respective Ub mutants, its expression was only reduced in the presence of UbWT (Fig. 2C, Input, lane 2). Immunoprecipitation with anti-HA revealed that the polyubiquitination of K130 was detected with UbWT, but not UbKO, further confirming that K130 is the target for ubiquitination (IP, lanes 2, 10). Notably, although this SERINC5-130K mutant had a similar level of polyubiquitination with UbK6R, UbK11R, UbK27R, UbK29R, and UbK63R as UbWT (IP, lanes 3–6, 9), this level was significantly decreased with UbK33R and Ub48R (IP, lanes 7–8).
To confirm the important role of K33 and K48 in SERINC5 polyubiquitination, we created another seven ubiquitin mutants, UbK6, UbK11, UbK27, UbK29, UbK33, UbK48, and UbK63, that only express each of those seven lysine residues of ubiquitin. We then repeated this experiment by expressing the SERINC5-130K mutant with these ubiquitin mutants. A similar level of SERINC5 polyubiquitination was detected from UbWT, UbK33, and UbK48 (Fig. 2D, IP, lanes 2, 7, 8), but this level was significantly reduced from UbK6, UbK11, UbK27, UbK29, UbK33, UbK63, and UbKO (IP, lanes 3–6, 9, 10). These results demonstrate that SERINC5 is preferably polyubiquitinated at K130 via K33 and K48-linked ubiquitin chains.
To confirm that CRL3KLHL20 plays a role in the K130 polyubiquitination, the SERINC5-K130 mutant was expressed with UbWT, UbK33, UbK48, and UbKO in the presence or absence of KLHL20-KD in HEK293T cells, and the K130 polyubiquitination state was determined exactly as described above. Again, we detected a similar level of SERINC5 polyubiquitination from UbWT, UbK33, and UbK48, and did not detect this polyubiquitination from UbKO (Fig. 2E, IP, lanes 2, 4, 6, 8). Importantly, KLHL20-KD completely disrupted this SERINC5 polyubiquitination (IP, lanes, 3, 5, 7). These results demonstrate that CRL3KLHL20 is responsible for the K130 polyubiquitination via K33 and K48-linked ubiquitin chains.
K130 is required for SERINC5’s localization to the plasma membrane. To understand how our lysine mutations affect SERINC5 subcellular localization, first, we tracked SERINC5 subcellular localization by confocal microscopy. SERINC5 was fused with a GFP tag at its C-terminus and similar lysine mutations were introduced into this SERINC5-GFP fusion protein. When these proteins were expressed in HeLa cells, WT SERINC5-GFP, 1-7K/R, and K179R were localized to the cell surface, whereas 1-14K/R, 1-19K/R, 1-11K/R, and K130R were found in the cytoplasm (Fig. 3A, HeLa, top panels). The 15-19K/R mutant was localized to both the cell surface and the cytoplasm. The colocalization of WT SERINC5-GFP, 1-7K/R, 15-19K/R, and K179R with DiIC18(5), confirmed that these proteins localize to the plasma membrane (Fig. 3A, HeLa, bottom panels). When WT SERINC5-GFP, K130R, and 1-19K/R were expressed in human Jurkat T cells, only the WT protein was found on the cell surface, consistent with the results observed from HeLa cells (Fig. 3A, Jurkat).
Next, we analyzed SERINC5 expression on the cell surface by flow cytometry. A FLAG-tag was inserted into the SERINC5 extracellular loop 3 region and similar lysine mutations were introduced into this SERINC5-iFLAG protein. These proteins were expressed in HEK293T cells, and their expression on the cell surface was determined via staining with anti-FLAG. WT SERINC5-iFLAG, 1-7K/R, 15-19K/R, and K179R were detected at much higher levels than 1-11K/R, 1-14K/R, 1-19K/R, and K130R (Fig. 3B). When this experiment was repeated in Jurkat cells, the poor expression of 1-19K/R and K130R was confirmed (Fig. 3B). These results are consistent with those from confocal microscopy.
Finally, we purified plasma membranes from cells to detect SERINC5 by WB. SERINC5 and its lysine mutants were expressed in HEK293T cells, and the plasma membrane fraction was isolated. CD4, a cell surface protein that is associated with the plasma membrane, was included as a control. Although WT SERINC5, 1-19K/R, K130R, K179R, and CD4 were detected at a similar level in total cell lysate (Fig. 3C, total lysate), only WT SERINC5, K179R, and CD4 were found in the membrane fraction, whereas 1-19K/R and K130R were not (Fig. 3C, membrane). Collectively, these results demonstrate that K130 determines SERINC5’s localization to the plasma membrane in a cell-type independent manner.
Polyubiquitination is required for SERINC5’s localization to the plasma membrane. Although Cul3 and KLHL20 decreased SERINC5 expression in the presence of ectopic ubiquitin (Fig. 1A), their ectopic expression and knockdown did not affect SERINC5 expression at steady state (Fig. 4A). We then determined how Cul3 and KLHL20 affect SERINC5 subcellular localization by using the similar approaches. Notably, knockdown of Cul3 and KLHL20 expression by CRISPR/Cas9 in HeLa and Jurkat cells disrupted SERINC5 expression on the cell surface when detected by confocal microscopy (Fig. 4B) and flow cytometry (Fig. 4C, lanes 4, 6, 11, 13), and this cell surface expression was restored by ectopic Cul3 or KLHL20 expression (Fig. 4B; Fig. 4C, lanes 5, 7, 12, 14). In addition, these KDs also significantly reduced the SERINC5 levels in the plasma membrane fraction (Fig. 4D, lanes 4, 6), which was also restored by their ectopic expression (lanes 5, 7).
We detected SERINC5 interaction with ubiquitin in live cells via bimolecular fluorescence complementation (BiFC)14, 15, 16. In this assay, a basic yellow fluorescent protein Venus was divided into N-terminal (VN) and C-terminal (VC) fragments. HA-tagged VN and FLAG-tagged VC were fused to the C-terminus of ubiquitin or SERINC5, respectively. When Ub-VN and SERINC5-VC were co-expressed in HeLa cells, green fluorescence signals were detected, indicating that ubiquitin-SERINC5 interaction occurred (Fig. 4E). These BiFC signals co-localized with SERINC5, confirming the specificity of this interaction. Importantly, these signals primarily localized to the cell surface, consistent with the conclusion that ubiquitination is required for SERINC5’s localization to the plasma membrane.
Next, the same lysine mutants were generated from SERINC5-VC and their interaction with Ub-VN was tested as above. BiFC signals were detected in cells expressing 15-19K/R, 1-7K/R, and K179R, but not 1-14K/R, 1-19K/R, 1-11K/R, 8-11K/R, 8-9K/R, and K130R (Fig. 4E). In addition, these signals primarily localized to the cell surface. Collectively, these results confirm the important role of polyubiquitination in SERINC5’s localization to the plasma membrane.
Polyubiquitination is required for SERINC5 downregulation by HIV-1 Nef. To understand how SERINC5 polyubiquitination affects its antagonism by Nef, we expressed SERINC5 and its lysine mutants with Nef in HEK293T cells and analyzed SERINC5 downregulation by WB. Initially, we analyzed 9 lysine mutants and found that 1-14K/R, 1-19K/R, 1-11K/R, 7-11K/R, 7-14K/R, 8-9K/R, and 8-11K/R were resistant to Nef (Fig. 5A, lanes 3–4, 7–8, 13–16, 19–20, 23–24, 31–34), whereas 15-19K/R, 1-7K/R, and 11-14K/R were as sensitive to Nef as the WT SERINC5 (lanes 5–6, 11–12, 21–22). These results suggested that K8 and K9 residues should be required for SERINC5 downregulation by Nef. We then tested K130R and K179R and found that K179R was still sensitive, whereas K130R became resistant to Nef (lanes 27–30). These results demonstrate that K130 determines SERINC5 sensitivity to Nef.
Previously, we reported that ectopic ubiquitin synergizes the SERINC5 downregulation by Nef 16. Therefore, we determined how these SERINC5 lysine residues affect such synergy. When SERINC5 proteins were expressed with Nef and ubiquitin in HEK293T cells, ubiquitin strongly promoted Nef downregulation of WT SERINC5 (Fig. 5B, lanes 1–4, 13–16). This ubiquitin synergy was also detected from Nef downregulation of the K179R mutant (lanes 21–24), but not the mutants containing the K130R mutation, such as mutants 1-19K/R, 8-11K/R, and K130R (lanes 5–12, 17–20). These results further confirm that K130 is critical for SERINC5 downregulation by Nef.
To understand how K130 determines the SERINC5 sensitivity to Nef, we analyzed the SERINC5-Nef interaction in live cells via BiFC as we and others reported16, 24, 25. When HA-tagged VN and FLAG-tagged VC were fused to the C-terminus of SERINC5 or Nef and expressed in HeLa cells, BiFC signals were detected, and co-localized with SERINC5, indicating an association of SERINC5-Nef in these cells (Fig. 5C). In addition, these signals were primarily detected in the cytoplasm, confirming the Nef-mediated downregulation of SERINC5 from the cell surface. When lysine mutations were introduced into SERINC5-VN, Nef interacted with 15-19K/R, 1-7K/R, and K179R, but not 1-14K/R, 1-19K/R, 1-11K/R, 8-11K/R, 8-9K/R, and K130R (Fig. 5C). These results demonstrate that K130 is required for SERINC5 interaction with Nef in cells and suggest that SERINC5 polyubiquitination plays an indispensable role in this interaction.
Polyubiquitination is required for SERINC5 anti-HIV-1 activity. To understand how lysine residues affect SERINC5 incorporation into virions, WT and ∆Nef HIV-1 were produced from HEK293T cells in the presence of SERINC5 and its lysine mutants. Virions were purified from culture supernatants by ultracentrifugation and analyzed by WB. We again confirmed that Nef downregulates 1-7K/R, 15-19K/R, and K179R, but not 1-11K/R, 1-14K/R, 1-19K/R, and K130R in cells (Fig. 6A). Consistently, the 1-7K/R, 15-19K/R, and K179R mutants were detected in virions, and their incorporation was inhibited by Nef. In contrast, none of the 1-11K/R, 1-14K/R, 1-19K/R, and K130R mutants were detected in virions. Thus, K130 is required for SERINC5 incorporation into virions, which confirms its role in SERINC5 expression on the cell surface.
To further confirm the important role of polyubiquitination in SERINC5 antagonism by Nef, SERINC5 was expressed with HIV-1 Nef in HEK293T cells in the presence of KLHL20- or Cul3-KD by CRISPR/Cas9, and/or their ectopic expression. Nef effectively decreased SERINC5 expression (Fig. 6B, lanes 1–2), a phenotype which was further enhanced by ectopic KLHL20 and Cul3 expression (lanes 3–4). This phenotype was disrupted by KLHL20-KD and Cul3-KD (lanes 6, 9), and recovered upon complementation with their ectopic expression (lanes 7, 10). Thus, Nef downregulation of SERINC5 is dependent on Cul3-KLHL20 mediated SERINC5 polyubiquitination.
Finally, we determined how K130 and polyubiquitination affects the SERINC5 anti-HIV-1 activity. Initially, ∆Nef HIV-1 was produced from HEK293T and Jurkat cells in the presence of SERINC5 and its lysine mutants, and viral infectivity was analyzed after infection of HIV-1 luciferase-reporter TZM-bI cells. 1-7K/R, 15-19K/R, and K179R inhibited the viral replication as strongly as WT SERINC5, whereas 1-11K/R, 1-14K/R, 1-19K/R, and K130R did not in both cell lines (Fig. 6C). Next, ∆Nef HIV-1 was also produced from HEK293T and Jurkat cells in the presence of SERINC5 and KLHL20- or Cul3-KD, and viral infectivity was analyzed again as above. Both KLHL20- and Cul3-KD disrupted the SERINC5 antiviral activity in both cell lines, a phenotype which was restored upon complementation with their ectopic expression (Fig. 6D). Thus, K130 and polyubiquitination are required for SERINC5 anti-HIV-1 activity.