The role of the SLP65 N terminus in the membrane targeting of SLP65

The N-terminal region of SLP65 is characterized by a high abundance of positively charged and hydrophobic amino acids. This region is remarkably conserved between species, with more than 90% sequence identity at the protein level. The profoundly conserved nature of the SLP65 N terminus is indicative of its structural and/or functional importance.

Indeed, as it was first demonstrated by(Kohler et al., in 2005,)the N terminus is essential for the function and membrane targeting of SLP65. The studies by my colleagues and me have confirmed that the N terminus is essential for the membrane association of SLP65, but our investigations point to a different molecular mechanism than that originally proposed by Koehler et al. A substitution of the residue I25, central to the proposed motif, with other aliphatic amino acids did not interfere with SLP65 function; excluding a functional leucine zipper (see Figure 4.9). Moreover, substitutions of those hydrophobic residues not corresponding to the heptad repeat of the leucine zipper, such as M21, V22, M33 and I36 to A and G, resulted in a strongly diminished Ca²⁺ flux and defective plasma membrane recruitment of SLP65 in DT40 cells (see Figure 4.23). This indicates a general requirement for the hydrophobic environment in the SLP65 N terminus.

My results point to a function of the SLP65 N terminus in membrane association via direct lipid binding. It was previously found in our group that the N terminus of SLP65 can bind to PIP-spotted membranes (PIP strips). It was hypothesized that this part of SLP65 constitutes a PIP-binding motif, which interacts with negatively charged lipids via electrostatic interactions (Herrmann, 2009). To confirm this finding, I used liposome floatation assays. In this method, PIPs are incorporated into a lipid bilayer, which provides a more “native” binding interface. I found that the SLP65 N terminus binds neutral liposomes independent of PIPs. Exchange of L18 to K abolished this interaction (see Figure 4.13 C), indicating the importance of hydrophobic residues in mediating lipid binding. This has been further confirmed by NMR analysis of the isolated N terminus (SLP65_5-50) in the presence of neutral liposomes. This analysis has identified the residues important for the liposome association – among them L18 (see Figure 4.21 C).

Together with my colleagues, Dr. M. Engelke and J. Kühn, we found that the N terminus confers membrane association to SLP65 already in resting DT40 cells, prior to any BCR stimulation. Constitutive association of the N terminus with membranes was already reported by Kohler et al.. However, they reported that SLP65 associates with the plasma membrane, and we, in contrast, argue that the vesicular membranes are targeted by SLP65 in resting cells. Detailed microscopic analysis revealed that the association of SLP65 with vesicles is reversible, as evident from the high fluorescence recovery rate upon photobleaching of vesicle-resident Citrine-tagged SLP65 (Figure 4.11). The cytosolic pool of SLP65 is also larger than the vesicular pool, as can be observed from fractionation studies and microscopic analysis. These observations indicate that the vesicle-SLP65 association in resting cells is weak. This is consistent with the results of the liposome floatation experiments, which show that the affinity of the N terminus for lipids is not very high, at least when neutral lipid species are concerned. The intrinsic disorder and the lack of structural transition in the N terminus upon lipid binding further support the hypothesis that the SLP65-vesicle association is mediated by weak hydrophobic interactions.

However, even this weak association seems to be critical for signaling, since the inactivation of the N terminus by mutations that shift the equilibrium towards the cytosolic fraction results in a signaling-incompetent SLP65, which cannot translocate to the plasma membrane and initiate the release of intracellular Ca²⁺ (see Figure 4.9 and 4.23). The vesicle residence of SLP65 in resting cells therefore appears to help the protein to get to the plasma membrane upon BCR stimulation. The easiest explanation for this observation is the use of vesicular transport by SLP65. Indeed, interference with vesicular trafficking by means of primaquine, a substance that inhibits budding of vesicles from donor membranes (Hiebsch et al., 1991), led to the inhibition of BCR signaling in DT40 cells (Engelke et al., 2014). Our studies therefore point towards a mechanism where the N terminus mediates association of SLP65 with vesicles in resting cells, which are transported to the plasma membrane and deliver SLP65 to the BCR upon antigen binding (see Figure 5.1).

Figure 5.1 The model of SLP65 translocation to the plasma membrane on intracellular vesicles.

The N terminus mediates the association of SLP65 with exosome-like vesicles in resting B cells.

The vesicles carry the SNARE protein Vamp7 and are positive for ATP (detectable by quinacrine-staining). Vesicular transport delivers SLP65 to the BCR upon activation.

The vesicular route might not be the only mechanism for the SLP65 membrane translocation in B cells. Actually, my results indicate that in certain cases SLP65 can be recruited to the plasma membrane in an N terminus-independent manner. We investigated the vesicular residence of SLP65 in an immature B cell line DT40. Analysis of the subcellular localization of SLP65 in a mature human DG75 B cell line showed that the intracytoplasmic dot-like distribution of SLP65 is discernable, but not as prominent as in DT40 cells. Colocalization experiments with quinacrine have indicated that these intracellular speckles indeed represent the vesicular population of SLP65 (data not shown). However, the phenotype of the N terminus inactivation via deletion or amino acid exchanges was not as strong in DG75 cells as in DT40 cells. All single point substitution variants of SLP65 mounted an only slightly diminished Ca²⁺ response upon BCR stimulation even though the plasma membrane recruitment of SLP65 was compromised (see Figure 4.24). Similarly, slp65^-/- mouse primary cells reconstituted with the ΔN and L18K variants of SLP65 mounted almost normal Ca²⁺ responses (see Figure 4.25). From

BCR

α β Ag

Syk

SLP65 ATP⁺

Y Y Y + SH2

developmental stages and the mechanism of SLP65 translocation via the vesicular route is more important for immature than for mature B cells. This is consistent with the finding by Koehler et al, who showed that the N terminus of SLP65 is essential for the pre-BCR signaling and for the developmental transition from the pre-B cell to the immature B cell stage (Kohler et al., 2005). The minor dependence of mature B cell types on the SLP65 N terminus implies that an alternative pathway regulates the membrane targeting of SLP65.

It is possible that a regulator, expressed in a developmental stage-specific manner, inhibits the vesicular pathway or enhances alternative pathways of SLP65 recruitment to the plasma membrane in mature B cells. This regulator could be a direct SLP65 interaction partner, or e.g. an enzyme modifying the lipid composition of the vesicles, making the attachment of SLP65 less/more likely. However, it is also possible that the differences in the requirement for the SLP65 N terminus are species-specific. In chicken, mouse and human there could be alternative molecules expressed which regulate the SLP65 membrane targeting.

5.3 The proposed mechanism used by the SLP65 N terminus for membrane