FAM21 contains multiple copies of a novel leucine-phenylalanine-acidic motif
FAM21 can be divided into two distinct regions: a “head” domain consisting of the N-terminal ~220 amino acids, which is responsible for incorporation into the SHRC, and a largely disordered “tail” containing the remaining ~1100 amino acids (). The FAM21 tail harbors at least 21 copies of a novel motif, which contains 3–10 acidic residues flanked by two hydrophobic Leu–Phe residues ( and Supplemental Figure S1). We term this the leucine-phenylalanine-acidic (LFa) motif. Leucine in the LFa motif is occasionally replaced with similar hydrophobic residues, such as isoleucine, valine, or methionine; some other residues, in particular serine, are frequently found within the acidic stretch. Enriched with hydrophobic and acidic residues, the LFa motif is reminiscent of sorting motifs that are recognized by adapter proteins and Golgi-localized, γ-ear-containing, Arf-binding (GGA) proteins (Bonifacino and Traub, 2003
). However, FAM21 is not a cargo protein, and, to our knowledge, the LFa motif is distinct from known protein–protein interaction motifs.
FIGURE 1: FAM21 directly interacts with retromer CSC. (A) Schematic representation of FAM21 constructs used. FAM21 contains 21 copies of the L-F-[D/E]3-10-L-F motif (LFa motif; black boxes). FN, FM, and FC contain repeats 1–6, 7–14, and 15–21, (more ...)
FAM21 directly interacts with retromer CSC
We previously showed that the FAM21 tail was important for endosomal localization of SHRC (Gomez and Billadeau, 2009
). To learn how FAM21 is targeted to endosomes, we took an unbiased approach to identify binding partners of the FAM21 C-terminus. We engineered a fragment encompassing LFa repeats 1–6 of FAM21 (FN) and another containing repeats 15–21 (FC; ). The 19-residue CapZ-binding motif of FAM21 (Jia et al., 2010
) was removed from FC. The two FAM21 fragments were expressed as glutathione S
-transferase (GST) fusions, immobilized, and incubated with bovine brain extracts. Three different bands were specifically retained by FN or FC fragments but not by GST control (Supplemental Figure S2). Mass spectrometry analysis revealed that they corresponded to adaptor protein 2 α2 (AP2α2), WAFL/FKBP15, and the retromer CSC component VPS35. The interactions of FAM21 with AP2α2 and WAFL/FKBP15 have been reported (Schmid et al., 2006
; Viklund et al., 2009
; Harbour et al., 2010
; Pan et al., 2010
), and we focused on the interaction with VPS35.
In cells VPS35 is constitutively associated with VPS26 and VPS29 to form the CSC (Hierro et al., 2007
). To determine whether the interaction between FAM21 and CSC was direct, we performed pull-down assays with purified recombinant proteins. Purified CSC was incubated with GST-FN, -FC, and -FM (a middle fragment of the FAM21 tail containing repeats 7–14). As shown in , all three GST-FAM21 fragments could efficiently pull down CSC. FC retained more CSC than FN and FM, and untagged FC could compete with FM for CSC binding (). These results demonstrate that different fragments of FAM21 can directly interact with a common site(s) on the CSC.
To confirm that the FAM21 tail is important for retromer binding in cells, we suppressed endogenous FAM21 and reexpressed the C-terminal tail containing the LFa motifs (R1-21) or the head domain involved in SHRC assembly (amino acids 1–356). Only FAM21 R1-21 was able to coimmunoprecipitate with endogenous VPS35, whereas the SHRC-interacting N-terminus did not (). Together, these data suggest that the FAM21 tail interacts with retromer.
Interaction between FAM21 and CSC is mediated by LFa motifs and VPS35
FN, FM, and FC can each bind the CSC. Yet these fragments have little similarity except for harboring multiple LFa motifs. Thus we examined whether the motifs could mediate interaction with CSC. We generated three additional FAM21 tail fragments: one is devoid of motifs (noR); the other two (R15-20 and R17-20) contain six and four repeats, respectively. Similar to the FC fragment, both R15-20 and R17-20 could effectively retain CSC. In contrast, the noR fragment failed to interact with CSC (). Thus CSC binding is likely mediated by the LFa motifs in the FAM21 tail.
FIGURE 2: Interaction between FAM21 and CSC requires the FAM21 motif and VPS35. (A) Schematic representation of FAM21 constructs used. (B) GST pull-down of retromer CSC using GST-FC and its derived fragments. Shown is a Coomassie blue–stained SDS–PAGE (more ...)
We next tested whether FAM21 could interact with an individual protein of the CSC or required the intact trimer. Individual VPS35, VPS26, and VPS29 proteins were used in pull-down assays with immobilized FC. Both VPS35 and the trimeric CSC, but not VPS26 or VPS29, were effectively retained by FC (). Therefore VPS35 is necessary and sufficient to interact with FAM21.
VPS35 regulates FAM21 and SHRC localization
We previously showed that FAM21 and SHRC colocalized with retromer and that suppression of FAM21 had no effect on the localization of retromer components (Gomez and Billadeau, 2009
). To determine whether retromer CSC regulates the localization of FAM21, we suppressed VPS35 using RNA interference (RNAi) and examined localization of FAM21 (Supplemental Figure S3). Consistent with a previous report (Harbour et al., 2012
), suppression of VPS35 resulted in significant dissociation of FAM21 from endosomal membranes and concomitant cytosolic dispersal of the protein. FAM21 localization could be rescued by reexpression of RNAi-resistant yellow fluorescent protein (YFP)–VPS35, which displayed strong colocalization with endogenous FAM21 (Supplemental Figure S3). Because FAM21 has the potential to target the entire SHRC to endosomes (Gomez and Billadeau, 2009
), we conclude that FAM21 and SHRC localization to retromer-enriched endosomal subdomains is VPS35 dependent.
Both the quality and the quantity of LFa repeats are important for interaction with retromer CSC in vitro
The FAM21 tail contains 21 LFa motifs, each with a distinct sequence (Supplemental Figure S1). Because the FC fragment demonstrated the most robust binding to VPS35, we used this region of FAM21 to better understand the LFa-CSC interaction. Specifically, we sought to learn which motifs could bind CSC most tightly, the stoichiometry of binding, and which residues of the LFa motifs contribute most significantly to binding. We tested several immobilized fragments derived from FC, consisting of one to seven repeats, for their ability to retain recombinant CSC (). We used the same total repeat concentration in each assay (rather than the same protein concentration), to normalize for the different number of repeats in each fragment. The retromer-binding ability of these FAM21 fragments varied substantially, with fragments bearing repeats 19, 20, or 21 interacting strongly and other fragments only weakly (if at all). Two different single-repeat constructs—R20 and R21—were able to retain CSC, but R17 was not. Similarly, the double-repeat fragments R19-20 and R20-21 retained significantly more CSC than any of the single-repeat fragments, but fragment R17-18 failed to retain any protein. Moreover, several of the larger fragments—namely R17-20, R17-21, and FC—retained CSC to an extent similar to the double-repeat fragments R19-20 and R20-21, whereas the triple-repeat fragment R17-19 retained a comparable amount of CSC to single-repeat R20 (). Thus, in solution-phase interactions (see Discussion) the final three repeats (19–21) make the most important contributions to binding the FC fragment. We note that repeats 19–21 are three of the four LFa repeats within FAM21 that contain a hydrophobic residue at the sixth position of the repeat (Supplemental Figure S1C), suggesting that this might be a feature that contributes to increased affinity for VPS35 (see later discussion of ).
FIGURE 3; Different FAM21 repeats show different efficiencies of CSC binding. (A) Schematic representation of FAM21 constructs used. (B) GST pull-down of retromer CSC using FC and its derived fragments. Shown is Coomassie blue–stained gel of input and bound (more ...)
FIGURE 6: All three elements of the FAM21 motif contribute to binding the CSC. (A) Schematic representation of FAM21 constructs used and degree of retromer binding determined by GST pull-down. (B) GST pull-down of retromer CSC using FAM21 R21 and its mutants 1–5. (more ...)
Next we used isothermal titration calorimetry (ITC) to quantify the interaction between FAM21 and CSC ( and Supplemental Figure S4). Different FAM21-derived fragments were titrated into CSC. ITC experiments showed that R21 binds CSC with Kd of 16.6 μM and ~1:1 stoichiometry. Moreover, titrations of the double-repeat fragments R19-20 and R20-21, when fitted to a model that assumes identical repeats within a fragment, showed that these fragments can bind to CSC in a ~2:1 manner (1/N = 2.56 and 2.04 for R19-20 and R20-21, respectively). These data yielded apparent Kd values of 6.8 and 3.2 μM for each site in R19-20 and R20-21, respectively (although these values should be viewed as approximate, as described in Materials and Methods). The data for the FC fragment can also be fitted to a single Kd of 2.4 μM for each site and stoichiometry of 2.38:1 (1/N = 2.38; stoichiometry for a system with seven potential binding sites should be viewed as highly approximate), supporting the notion that in solution the interactions between FC and retromer CSC are mediated primarily by the last three repeats, which exhibit high affinity for CSC. Due to the stoichiometry of the interaction, it is likely better to titrate CSC into Fam21 fragments; however, the high concentration of CSC required (at least 200 μM) makes such an experiment unfeasible.
Activities of FAM21 tail constructs.
Finally, ITC data on the FN fragment, which contains six repeats predicted to have low affinity for CSC, could be fitted to a single Kd of 11.5 μM for each site and stoichiometry of ~3:1. This affinity is lower than that of the FC fragment, in agreement with our pull-down results (). Taken together, our biochemical data indicate that the LFa motif is the minimal CSC-binding element but that different motifs have quite different affinities for CSC. As described in the Discussion, it is likely that when retromers are bound to membranes, avidity effects involving both high-affinity and low-affinity sites in the FAM21 tail will afford high overall affinity of the SHRC for membranes.
Both the quality and the quantity of FAM21 repeats are important for its endosomal localization
We next determined how the number and quality of LFa repeats collectively contribute to endosomal localization of FAM21. We used a suppression/reexpression system to deplete endogenous FAM21 and reexpress YFP-tagged FAM21 truncation mutants ( and ). As we showed previously (Gomez and Billadeau, 2009
), wild-type YFP-FAM21 had a punctate localization juxtaposed with the early endosome marker EEA1 (). Similarly, FAM21 retained robust endosomal localization when up to five repeats were deleted from the N-terminus of FAM21(R5-21
). However, when we made additional N-terminal deletions (R6-21
), the colocalization of FAM21 mutants with EEA1 diminished appreciably, although not completely. Finally, FAM21 was redistributed from endosome membranes to the cytosol when 10 repeats were deleted from the N-terminus (R11-21
). Consistent with the importance of R20
in binding retromer in vitro, we found that FAM21 became cytosolic with deletion of both C-terminal repeats (R5-19
), and deletion of only R21
substantially diminished FAM21 localization (R5-20
). Last, deletion of R11-14
in the context of R5-21
) also resulted in considerably diminished endosomal localization. These experiments suggest that the endosomal localization of FAM21 requires a combination of multiple repeats of high and low affinity. Therefore both the number of FAM21 repeats (which will likely afford avidity when retromer is at membranes; see later discussion) and the exact repeat sequence (which determines the affinity of any particular repeat) collectively contribute to the FAM21 distribution between cytosol and endosomal membranes.
FIGURE 4: Number of the FAM21 repeat dictates its endosomal localization. (A) Schematic representation of YFP-fused FAM21 truncation mutants and degree of early endosomal (EE) localization. (B) HeLa cells were transfected with control vector (YFP only) or various (more ...)
FIGURE 5: Quality of FAM21 repeat also dictates endosomal localization. (A) Schematic representation of YFP-fused FAM21 truncation mutants and degree of early endosomal (EE) localization. (B) HeLa cells were transfected with control vector (YFP only) or various (more ...)
Because R5-21 but not R5-19 can localize to early endosomes ( and ), we tested whether addition of the four N-terminal repeat motifs onto R5-19 (R1-19) would affect localization. Importantly, R1-19 partially rescued endosomal localization (). Thus the addition of lower-affinity repeats to the N-terminus can partially rescue FAM21 localization, confirming that a combination of low-affinity interactions can support endosomal targeting. Interestingly, although FC can efficiently bind retromer CSC in vitro, a YFP-tagged FAM21-FC* (asterisk indicates that it contains the CAPZ-binding motif) construct was unable to localize to endosomal membranes, suggesting that the high-affinity binding repeat motifs found in this region are insufficient to cause membrane targeting (). To test whether the number of repeats could affect subcellular localization, we created a fusion protein of FC* and R11-21 fragments of FAM21, which each individually does not localize to endomembranes. Significantly, this fusion protein (FC*/R11-21) relocalized to EEA1+ endosomes, consistent with the idea that both the number and the strength of retromer CSC-binding interactions contribute to FAM21 targeting to endosomes (). Thus, varying the LFa motif number and sequence can shift the distribution of FAM21 between endosomes and cytosol.
Mutagenesis of the FAM21 repeat sequence
The LFa motif comprises three elements: two Leu–Phe or Ile–Phe dipeptides and one acidic stretch. We mutated each of the three elements in the context of the R21 repeat and tested the interaction with CSC (). As shown in , wild-type R21 retained CSC on the solid support. However, when the first IF dipeptide motif was mutated to AA (mt1) the interaction with CSC was abolished; the single F-to-A mutant (mt2) behaved similarly. In contrast, mutation of F to A (mt5) in the second dipeptide element only weakly affected binding. Changing the central residues from DD to KK (mt3) or PL to DD (mt4) also dramatically decreased CSC binding (). Last, a single L-to-D mutation (mt6) of the hydrophobic residue at position 6 resulted in a significant loss of binding (). Similar conclusions are supported by mutagenesis of double-repeat FAM21 fragments (Supplemental Figure S5, A–E). Taken together, these data demonstrate that the LF/IF dipeptide motifs, as well as the acidic charged region, are critical to CSC interaction and support the notion that the hydrophobic residue at position 6, which is found in R19-R21, may contribute to increased affinity for VPS35.