Feline Lindhout

114 4 recognizing the luminal side of SV membrane protein Syt, while neurons were briefly stimulated by bicuculline. Subsequently, neurons were fixed and the fluorescence intensity of internalized Syt at individual presynaptic boutons was measured. VAP was depleted from neurons by expressing shRNAs targeting VAPA and VAPB, which we have validated in the previous studies (Teuling et al, 2007; Kuijpers et al, 2013). Co-depletion of VAPA and VAPB showed a marked decrease (~ 50%) in Syt internalization compared to control cells (Fig 1C,D). In addition, VAPA and VAPB knockdown resulted in a slight decrease in bouton size and bouton density (Fig 1C, S1B,C). In summary, we observed that loss of function of ER proteins VAPA and VAPB was accompanied by decreased SV cycling and defects in bouton maintenance. VAPA and VAPB associate with brain-enriched SCRN1 proteins VAPs function as ER receptors for a large number of VAP-associated proteins containing a FFAT or FFAT-like motif (Murphy & Levine, 2016). To gain more insight into the underlying mechanism of VAP at presynaptic sites, we sought to identify the VAP interactor(s) that could be involved in controlling this function. In a recent study, many new VAP-associated proteins were identified by pull-down and mass spectrometry analysis, including the cytoplasmic protein SCRN1 (Murphy & Levine, 2016). Western blot analysis of lysates from different rat tissues using two different antibodies revealed that SCRN1 is abundantly enriched in brain tissues (Fig 1E, S1D). This is consistent with the reported enriched expression of SCRN1 in the brain as described in various online expression databases (Protein Atlas, Expression Atlas, Alan BrainAtlas). We confirmed the association between VAP and SCRN1 with various biochemical assays. First, we conducted a pull-down experiment on lysates of HEK293T cells co-expressing biotinylated GFP (BioGFP) or GFP-SCRN1 (BioGFP-SCRN1) and Myc- VAPAor Myc-VAPB. Both Myc-VAPAand Myc-VAPB efficiently co-precipitated with BioGFP- SCRN1 (Fig 1F, G). Next, we examined the SCRN1 interactome using a more unbiased approach and performed BioGFP-SCRN1 pull-downs followed by mass spectrometry analysis using HEK293T cell lysates and adult rat brain extracts. The associations between SCRN1 and the VAPs were identified in both HEK293T lysates and brain extracts (Fig 1H, S1E). Of all potential SCRN1-interacting proteins, both VAPA and VAPB showed the highest peptide-spectrum match (PSM) values in both datasets. Together, these biochemical data indicated that VAPs are associated with SCRN1. VAP-associated protein SCRN1 is involved in modulating SV cycling We next tested whether SCRN1 was present at presynaptic sites and if this protein could be engaged in modulating SV cycling. Similar to VAP, immunostaining for endogenous SCRN1 revealed a punctate pattern throughout the neuron and regularly co-localized with presynaptic marker Syt (Fig 1I). Exogenous GFP-SCRN1 showed a diffuse cytoplasmic signal, which also co-localized with presynaptic boutons (Fig S1F). To conduct loss-of- function experiments, we next generated and validated three shRNA targeting SCRN1 and continued our depletion experiments with a single shRNA (Fig S1G–J). We tested the role of