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“TMEM16K is an interorganelle regulator of endosomal sorting”

Communication between organelles is essential for their cellular homeostasis. Neurodegeneration reflects the declining ability of neurons to maintain cellular homeostasis over a lifetime1. The endolysosomal pathway plays a prominent role in this process by regulating protein and lipid sorting and degradation2. Here, we report that TMEM16K, an endoplasmic reticulum lipid scramblase3 causative for spinocerebellar ataxia (SCAR10), is an interorganelle regulator of the endolysosomal pathway. We identify endosomal transport as a major functional cluster of TMEM16K in proximity biotinylation proteomics analyses. TMEM16K forms contact sites with endosomes, reconstituting split-GFP with small GTPase RAB7. Our study further implicates TMEM16K lipid scrambling activity in endosomal sorting at these sites. Loss of TMEM16K function led to impaired endosomal retrograde transport and neuromuscular function, one of the symptoms of SCAR10. Thus, TMEM16K-containing ER-endosome contact sites represent clinically relevant platforms for regulating endosomal sorting.

frequently associated with their functions as calcium activated chloride channels or phospholipid scramblases, their yeast homolog Ist2 was one of the first reported MCS proteins, playing a vital role in lipid homeostasis at MCS between the ER and plasma membrane 13 . Here we focus on TMEM16K, the mammalian family member most closely related to Ist2 (Supplemental Fig. 1a) and causative of a recessive form of spinocerebellar ataxia (SCAR10) 14 .
We generated mouse models with either ubiquitous or neuron specific loss of TMEM16K (Supplemental Fig. 1b) to evaluate if the pathology is conserved between mouse and human. As impairment of neuromuscular function is a classical symptom of ataxia, we analyzed neuromuscular junctions (NMJ) 15 in TMEM16K knockout mice at 6 and 24 months of age. Using bungarotoxin staining, we found a progressive reduction in the size of the NMJ (Fig. 1a,b).
Moreover, knockout mice displayed increasing hindlimb clasping, a behavioral phenotype marking disease progression in a number of mouse models of neurodegeneration 16,17 (Fig. 1c, Supplemental Video 1). As TMEM16K is broadly expressed (Supplemental Fig. 1c), we analyzed neuron specific TMEM16K knockout mice and wild type littermates at 24 months of age to evaluate whether loss of TMEM16K in neurons is sufficient to cause the observed phenotypes.
These animals lacking neuronal TMEM16K displayed increased hindlimb clasping, as well as an impaired ability to complete a ledge-walking test (Fig. 1d). Together, these results demonstrate a phenotypic linkage between loss of TMEM16K and impaired neuromuscular function that is conserved between mice and human.
TMEM16K has previously found to be localized to the endoplasmic reticulum 3 , a localization shared with its yeast 18 and Drosophila 19 homologs. Consistent with these studies, we found TMEM16K localizes to the endoplasmic reticulum (ER) as evident by colocalization with several established endoplasmic reticulum markers (Fig. 1e,f, Supplemental Video 2).
To elucidate the potential cellular functions of TMEM16K in an unbiased manner, we set out to identify the TMEM16K protein interaction network using proximity-dependent biotinylation [20][21][22]. This approach uncovers direct and indirect interactions within a 10nanometer range of the promiscuous biotinylation enzyme tagged to the protein of interest (Fig.   2a). We tagged TMEM16K with biotin ligase and confirmed retention of both endoplasmic reticulum localization (Supplemental Fig. 2a,b) and biotinylation activity (Supplemental Fig. 2c), permitting the identification of TMEM16K-proximal proteins by mass spectrometry of affinitypurified biotinylated proteins from transfected cells (Fig. 2a). We obtained a list of potential TMEM16K interactors (Fig. 2b, Supplemental Table 1) and, instead of hand-picking a few candidates, we visualized this list as a protein-protein interaction network to identify the most biologically interconnected clusters of proteins which could infer TMEM16 function (Fig. 2b). First, we calculated protein-protein interaction enrichment to determine if the obtained candidate list has more or less interactions among themselves, as compared to a random set of proteins of similar size. Protein-protein interaction enrichment p-value of the TMEM16K network is p<1.0E- 16, suggesting biological connection of proteins that interact with TMEM16K. Next, we performed functional enrichment analysis, and overlaid the major functional categories on our candidates, suggesting the presence of functional clusters in our candidate list. Hence, we performed clustering analysis to bioinformatically identify such clusters, defined as highly interconnected nodes, and generated a simplified network of TMEM16K major clusters overlaid with functional enrichment categories (Fig. 2c). As expected, when evaluating a protein over its lifetime, we found clusters involved in protein processing and degradation. Consistent with the function of its Drosophila homolog 19 , we also identified a cluster of proteins involved in nuclear organization.
Unexpectedly, this analysis revealed that endosomal transport, in particular endosomal retrograde trafficking, is a major cluster in the TMEM16K network (Fig. 2c).
Dysfunctions of endosomal transport are tightly associated with neurodegenerative diseases 23,24 . As the TMEM16K interactome pointed to endosomal retrograde transport, we investigated whether TMEM16K is required for proper trafficking of the cation-independent mannose-6-phosphate receptor (CI-MPR), which is the best-studied retrograde-transport cargo in mammals 25,26 . In wild type primary mouse embryonic fibroblasts (MEF), when an antibody that recognizes the extracellularly exposed CI-MPR is pulse chased from the plasma membrane, it gets internalized in the endosomes and subsequently transported through the endosomal retrograde pathway to the perinuclear region corresponding to the trans-Golgi network (TGN) within 60 min 1,2 (Fig. 3a). However, in MEF from TMEM16K knockout mice, the internalized antibody against CI-MPR remained dispersed peripherally during the 60 min period of pulse chase, consistent with a defect in endosome to trans-Golgi retrograde trafficking (Fig. 3a).
Reintroduction of TMEM16K rescued this CI-MPR retrograde trafficking defect of mutant MEF (Fig. 3a). As this pathway is also co-opted by a subgroup of pathogens during their entry into cells, we used cholera toxin B (CTxB) 27 to further corroborate our finding. Indeed, following CTxB internalization, TMEM16K knockout cells displayed reduced CTxB colocalization with the Golgi marker GM130 after 60 min of pulse chase, which can be rescued by reintroduction of TMEM16K ( Fig. 3b), confirming that TMEM16K is required for proper endosome to trans-Golgi retrograde trafficking. However, no change was observed in the localization of Golgi complex proteins, nor in the morphology of the Golgi complex ( Fig. 3b; Supplemental Fig. 3), suggesting that loss of TMEM16K function does not affect the Golgi complex. Altogether, these data demonstrate that depletion of the ER-resident protein TMEM16K perturbs endosomal retrograde trafficking, a defect similar to that observed upon depletion of known cargo-sorting components 26,28,29 .
To ensure that the observed defect in endosomal retrograde transport is not due to impaired anterograde secretory pathway, we have taken advantage of a novel approach that allows synchronization of protein transport through the biosynthetic pathway 30,31 . Using this RUSH system we tracked the biosynthetic transport of three transmembrane proteins with different steady state distributions: the glycosylphosphatidylinositol anchor (GPI; transported to plasma membrane), the transferrin receptor (TfR; transported to plasma membrane, early endosomes, and recycling endosomes), and the cation-dependent mannose-6-phosphate receptor (CD-MPR; transported from TGN directly to early/late endosomes). We found no difference in the transport through the biosynthetic pathway between TMEM16K wild type and knockout cells (Fig. 3c- showing that the anterograde secretory pathway is unaffected. To look for evidence for potential defects upstream of the endosomal retrograde sorting, we evaluated first whether endocytosis is affected by examining the internalization of fluorescently labelled transferrin. We found no differences between TMEM16K wild type and knockout cells (Fig. 3f). Next, we performed EGF pulse-chase experiments to further evaluate maturation from early to late endosomes. We found no difference between the wild type and knockout cells in the colocalization of EGF with the late endosomal marker Rab7 at the 10 min, 15 min and 40 min time points (Fig. 3g), indicating that the mutant phenotype arose from a defect at or after the Rab7 stage of endolysosomal maturation. These results show that the upstream endosomal pathway is unaffected in TMEM16K knockouts. However, at 60 min there was a larger fraction of EGF that was retained in Rab7 endosomes in TMEM16K knockout cells, compared to wild type cells (Fig.   3g), suggesting defect in endosomal sorting.
To evaluate whether endolysosomal pathway downstream of endosomal sorting was perturbed, we examined a major distinguishing feature of endolysosomal maturation, acidification.
Using Lysosensor Green DNP-189, a fluorescent pH indicator that partitions into acidic organelles, we found that acidification is impaired in the absence of TMEM16K (Fig. 3h). To confirm that the differences in DNP-189 fluorescence reflected differences in pH within organelles, we utilized the protonophore FCCP to selectively eliminate the pH gradient. Consistent with an inability to form and/or maintain a proton gradient, TMEM16K knockout cells displayed both a slower rate of FCCP-induced proton leak and an impaired ability to stabilize proton loss compared with wild type cells (Fig. 3i). To further test the TMEM16K involvement in the observed defect, we performed rescue experiments with wild type TMEM16K or mutant TMEM16K with substitutions of the conserved calcium binding acidic residues required for protein function (E448Q/D497N/E500Q/E529Q/D533N) 3,32,33 . We expressed wild type or mutant TMEM16K in primary cells from wild type or TMEM16K knockout mice. Transfecting mutant TMEM16K into wild type cells has dominant negative effect. Expression of wild type, but not mutant TMEM16K in primary cells lacking TMEM16K rescued the acidification defect, demonstrating that TMEM16K is required for normal maturation of endolysosomal compartments (Fig. 3j). Taken together, these results show that loss of TMEM16K causes a defect in endosomal retrograde sorting, and deficiencies within the later stages of the endolysosomal system.
The requirement of TMEM16K for endosomal retrograde trafficking raised the question how this endoplasmic reticulum localized protein affects endosomes. Given that proteomics revealed that TMEM16K is in the proximity of the endosomal compartment for direct or indirect interactions, we considered the possibility that TMEM16K facilitates endosomal sorting through interorganelle communication at the sites of contact between the ER and endosomes. Membrane contact sites between the ER and endosomes have been shown to increase as endosomes mature 34 , to define endosome fission 7 , and to control association of endosomes with the cytoskeleton 35 , all of which are essential for proper endolysosomal function. The TMEM16K proteomics dataset contained multiple proteins known to function at ER-endosomal contact sites including VAPA and VAPB 35-37 , SNX1 and SNX2 35 , Rab7A 34,37,38 , PTP1B 39 ( Fig. 2b; Supplemental Table 1), suggesting that TMEM16K acts at or in the proximity of these membrane contact sites.
Hence, we applied in cell culture the same proximity biotinylation approach labeling within a 10-nanometer range used for proteomics, in order to visualize TMEM16K-dependent labeling of Rab7-positive endosomes (Fig. 4a). We found that TMEM16K-proximity dependent labeling overlapped with endogenous Rab7. For a dynamic view, we performed live imaging of fluorescently labeled TMEM16K in the ER, with fluorescently labeled Rab7, and fluorescently conjugated EGF in endosomes (Supplemental Video 3, Fig. 4b). These experiments revealed highly dynamic movements of both compartments, as well as their contacts. Using structured illumination microscopy (SIM), we imaged TMEM16K and Rab7-positive endosomes, and visualized dually labeled ER-endosome contact sites (Fig. 4c-e). Next, we used the split-GFP system to specifically evaluate TMEM16K interorganelle contact sites. Split-GFP reconstitution has been extensively used to detect interorganelle contact sites 37-39 . TMEM16K was tagged with a GFP11 fragment, while several ER (VAPA, OSBPL8) and endosomal (Rab7, OSBPL9, OSBPL11, VPS26, VPS35, SNX1, SNX2) proteins were tagged with the GFP1-10 fragment. We selected proteins that were identified in the TMEM16K proteomics and implicated in ERendosomal MCS (VAPA, Rab7, OSBPL11, VPS26, SNX1, SNX2), as well as proteins that are not TMEM16K proteomics interaction candidates, but are known to participate in similar processes/compartments (OSBPL8, OSBPL9, VPS35) as negative controls. Since TMEM16K forms a dimer 3,32 , we validated the split-GFP approach by expressing TMEM16K-GFP1-10 and GFP11-TMEM16K to reconstitute the split-GFP (Fig. 4f). Reconstitution of split-GFP between TMEM16K and any of the tested candidates suggests that they directly interact, bringing the two GFP fragments to such close proximity that they can reconstitute the fluorescent GFP. Inability to reconstitute split-GFP suggests that TMEM16K and the tested candidate are not in close proximity, though we cannot exclude the possibility that steric hindrance may prevent the split-GFP reconstitution (Fig. 4g). Out of all the combinations tested, only Rab7 reconstituted split-GFP with TMEM16K, demonstrating that ER-localized TMEM16K forms contacts with Rab7 endosomes ( Fig. 4h; Supplemental Fig. 4). Rab7 is a GTPase that cycles between inactive GDP bound states and active GTP bound states. To evaluate further the specificity of TMEM16K interaction with the Rab7, we generated Rab7 mutants: constitutively active Q67L that mimics permanently GTP-bound Rab7 and inactive T22N that mimics permanently GDP-bound Rab7 43 .
We found that TMEM16K was able to reconstitute split-GFP only with the constitutively active Rab7 Q67L mutant ( Fig. 4i), but not with the inactive Rab7 T22N mutant (Fig. 4j), further validating the specificity of the observed contact between TMEM16K and Rab7 endosomes.
The yeast homolog Ist2 mediates ER-plasma membrane contact sites by directly binding plasma membrane specific phosphatidylinositol-(4, 5)-bisphosphate (PtdIns(4,5)P2) with its Cterminus 44 . The presence of a series of positively charged residues in the TMEM16K N-terminal cytosolic domain prompted us to hypothesize that, in addition to its interaction with endosomal proteins like Rab7, TMEM16K may directly bind phosphatidylinositols specific for the endolysosomal compartments. To test this hypothesis, we purified the N-terminal domains of TMEM16K and two other mammalian family members as control, TMEM16F and TMEM16A, to evaluate their lipid binding via a protein lipid overlay assay ( Fig. 5a, and found no perturbation in the absence of TMEM16K (Figure 6a). Likewise, there was no alteration of plasma membrane PtdIns(4,5)P2 visualized with the 2PH-PLCΔ-GFP biosensor or anti-PIP2 antibody (Supplemental Fig. 3). Furthermore, unlike VAPA/B DKO cells 35 , we found no perturbation of the actin cytoskeleton in cells lacking TMEM16K (Fig. 6a). However, by utilizing P40PX-EGFP to detect the distribution of PtdIns3P 50 , we found enlarged PtdIns3P vesicles in the absence of TMEM16K (Fig. 6b,d). Reintroducing TMEM16K in KO cells fully rescued the enlarged PtdIns3P vesicles phenotype (Fig. 6b,d). PtdIns3P is a precursor for the generation of PtdIns (3,5)P2 by the only mammalian PtdIns 5-kinase, PIKfyve 51 , which when perturbed also leads to enlarged endosomes. Thus, we wondered if this conversion is perturbed in the absence of TMEM16K. As we were not able to reliably visualize PtdIns(3,5)P2 with fluorescent reporter in primary cells from wild type and TMEM16K knockout mice, we used the pharmacological inhibitor of PIKfyve 52 , YM201636, and visualized its effects on PtdIns3P (Fig. 6c). Inhibiting PIKfyve in TMEM16K wild type cells recapitulated the TMEM16K knockout phenotype. However, in the TMEM16K knockout cells we did not observe additional cumulative effect, suggesting that conversion of PtdIns3P to PtdIns (3,5)P2 is impaired in the absence of TMEM16K (Fig. 6c,d).
Altogether, our results strongly indicate that, while there could be coordination of TMEM16K and VAPA/B MCS functions in mediating endosomal retrograde trafficking, TMEM16K affects endosomal sorting in a manner independent of the VAPA/B pathway.
To better define human mutations linked to spinocerebellar ataxia (SCAR10), we tested three known single amino acids missense mutations (Phe171Ser, Phe337Val and Asp615Asn; Fig. 6e,f) 3 . These human disease variants expressed normally and localized to the endoplasmic reticulum in heterologous cells (Supplemental Fig. 6). Out of these three, Phe171Ser was able to reconstitute split-GFP with Rab7 ( Fig. 6g). Remarkably, unlike wild type TMEM16K that was able to completely rescue the CI-MPR retrograde trafficking defect in TMEM16K knockout cells, these three human disease variants were unable to rescue (Fig. 6i,j). Thus, our results suggest that impairment of the endosomal pathway could be a contributing factor in the development of the SCAR10 pathology.
Given that some TMEM16K mutants can reconstitute split-GFP with Rab7, but are unable to rescue the CI-MPR retrograde trafficking defect in TMEM16K knockout cells (Fig. 5d, Fig. 6). When reintroduced into TMEM16K KO cells, the putative non-scramblase chimera TMEM16K-SCRD16A failed to reconstitute split-GFP with Rab7 (Fig 6h) and it could not rescue the retrograde trafficking defect as assayed by CI-MPR internalized antibody distribution ( Fig. 6i,j). However the putative scramblase chimera TMEM16K-SCRD16F was able to reconstitute split-GFP with Rab7 ( Fig. 6h) and rescue the endosomal retrograde trafficking defect ( Fig. 6i,j), suggesting that the lipid scrambling activity of TMEM16K could be required for endosomal sorting.
In conclusion, our study has discovered that TMEM16K acts as an interorganelle regulator of ER-endosomal communication at their sites of contact. Loss of TMEM16K leads to dysfunction of the endolysosomal pathway, which could be rescued by wild type TMEM16K but not mutant   their WT (7 animals       Immunocytochemistry to evaluate subcellular localization of TMEM16K mutants to endoplasmic reticulum. COS7 were co-transfected with ER-tdTomato labeling ER (pseudocolored in cyan) and TMEM16K mutant constructs tagged with FLAG or V5 tag, which was revealed with anti-FLAG or anti-V5 antibody, respectively (pseudocolored in magenta). Scale bar 10 µm.

Supplemental Video 1.
Representative hindlimb clasping of neuron specific TMEM16K WT and KO animal at 24 months of age.

Supplemental Video 2.
Live imaging of COS7 cells transfected with mClover3-TMEM16K (pseudocolored in magenta) and ER-tdTomato (pseudocolored in cyan). Video presents overlay of the two channels.
Nikon Imaging Center Library. We have obtained cDNA for VPS26A (Clone ID: BC022505) OSBPL9 (Clone ID: BC025978) and OSBPL11 (Clone ID: pCS6(BC065213)) from Transomics. Detailed description of all the constructs designed for this study is given in Supplemental Table 2. Site-directed mutagenesis was performed using PfuTurbo polymerase followed by DpnI digestion protocol. All other plasmids were constructed using classical subcloning or Gibson assembly using Hot Start High Fidelity Q5 polymerase from NEB. All constructs generated and used in this study were verified with sequencing of the coding region of the plasmids.

TMEM16K mice models
We obtained TMEM16K conditional knockout mice (Ano10tm1a(EUCOMM)Wtsi)) generated by the International Mouse Knockout Consortium and ordered from EMMA (EMMA ID: 08927 knockout littermates were kept gender segregated but genetically mixed with up to 5 animals per cage and aged up to 24 months.

Neuromuscular junction staining
Animals were anesthetizes with isofluorane, and perfused first with PBS and then with 4 % PFA. In all the animals, we have dissected, analyzed and compared the same muscle localized in the hindlimbs. After isolation, muscles were immersed in blocking buffer containing 0.5% Triton-X 100 and 10% donkey serum in PBS at 4°C overnight. The muscles were then washed three times with 1 x PBS at room temperature for 15 minutes each time followed by incubation with fluorescently labeled α-Bungarotoxin overnight at 4°C on a rotating platform. The muscles are again washed 3 times with 1 x PBS, and mounted Fluoromount-G medium (SouthernBiotech) with weight applied on the cover glass to obtain flat preparation suitable for imaging. NMJ were imaged on confocal microscope as z-stacks and represented as maximum intensity projections to ensure entire structure is captured, and then analyzed in Fiji Software (ImageJ, NIH).

Hindlimb clasping and ledge assays
Behavior of WT and TMEM16K KO littermates was analyzed using hindlimb clasping and ledge assays. Hindlimb clasping is a marker of disease progression in a number of mouse models of neurodegeneration 1 , while ledge assay is a direct measure of coordination, which is impaired in cerebellar ataxias and many other neurodegenerative disorders. The evaluation of mice was done as previously described 2 . Briefly, each measure is recorded on a scale of 0-3 depending on the severity of phenotype. For hindlimb clasping, mouse was lifted clear of all surrounding objects by grasping the tail near its base. The hindlimb position was observed for 1 min. If the hindlimbs were consistently splayed outward, away from the abdomen, it was assigned a score of 0. If one hindlimb retracted toward the abdomen for more than 50% of the time suspended, it received a score of 1. If both hindlimbs were partially retracted toward the abdomen for more than 50% of the time suspended, it received a score of 2. If its hindlimbs were entirely retracted and touching the abdomen for more than 50% of the time suspended, it received a score of 3. For the ledge assay, mouse was observed as it walked along the cage ledge and lowered itself into its cage. A wild type mouse walks along the ledge without losing its balance, and lowers itself back into the cage using its paws. This was assigned a score of 0. If the mouse lost its footing while walking along the ledge, it received a score of 1. If it did not effectively use its hind legs, or landed on its head rather than its paws when descending into the cage, it received a score of 2. If it fell off the ledge, while walking or attempting to lower itself, or shaked, it received a score of 3. All behavior analysis was done blinded of the mice genotype, with each mice tracked during the analysis with a random numeric code.

RNA extraction and RT-PCR
Animals were euthanized with CO2 and tissue was immediately dissected on ice. Total containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37ºC and 5% CO2.
We used HEK293 cells for proteomics due to their easy maintenance, suitability for scaling

Conventional microscopy
We performed majority of the fixed and live imaging on Leica SP8-X inverted confocal system equipped with HyD hybrid detectors, adaptive focus control and Okolab environmental control incubator cage. Live imaging was performed on Nikon Ti inverted fluorescence microscope with CSU-22 spinning disk confocal, EMCCD camera, and incubator, CO2, and humidity control at the UCSF Nikon Imaging Center. Images are represented using pseudocolors suitable for color-blind palette.

Evaluation of Golgi complex morphology
We analyzed Golgi complex morphology as previously reported 3 . Briefly, we performed immunochemistry on wild type and TMEM16K knockout primary cells with either antibody against TGN38 as trans-Golgi complex marker, or GM130 as cis-Golgi marker. Images were acquired on a Leica SP8-X confocal microscope, with a pinhole of 0.5 AU and voxel depth of 0.19 µm. Imaris software was used to reconstruct Golgi complex and quantify volume and area for each Golgi complex. Index of fragmentation is defined as ratio of volume and area.

Proximity Ligation Assay (PLA) for in situ detection of ER-endosome contacts
Mouse wild type and TMEM16K knockout fibroblasts were seeded on poly-L-lysine-coated 8-well chamber slide at density of 20 000 cells per chamber, fixed with 4% PFA for 10min, and subjected to proximity ligation assay according to manufacturer's protocol (SigmaAldrich, Duolink® In Situ Detection Reagents FarRed, #DUO92013). Briefly, after permeabilization with 1X PBS / 1% BSA / 0.3% Triton X-100 for 30min, the cells were subjected to blocking, incubation with mouse anti-VAPB and rabbit anti-Rab7 antibodies overnight at 4°C, hybridization with PLA probes, ligation, amplification, and mounted in Duolink mounting media with DAPI (SigmaAldrich, #DUO82040-5ML). Same procedure was done without inclusion of primary antibodies, as negative control to confirm the specificity of the observed PLA signal. Non-overlapping images were randomly acquired throughout the slide of each sample on the Leica SP8-X confocal microscope. The Fiji Software (ImageJ, NIH) was used to quantify the number of PLA puncta (https://fiji.sc) indicative of a close apposition between VAPB (ER) and Rab7 (endosome). The number of PLA puncta measured per image was expressed as ratio to number of nuclei in the same image, giving a measurement of the average number of puncta per cell.

Electron microscopy
Primary wild type and TMEM16K knockout cells were fixed with 4% paraformaldehyde (PFA) followed by post-fixation in EM fixative (2% PFA and 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH = 7.4) and processed for electron microscopy at the UCSF Veterans Affairs Medical Center Pathology Core. Electron micrographs were analyzed blinded, where endosomes and endoplasmic reticulum were separately identified and labeled using Fiji Software (ImageJ, NIH). The percentage of endosomes with an ER contact site were quantified as described previously 4 , where endosome-ER membrane contacts were defined as proximity >30 nm.

Structured illumination microscopy (SIM) super resolution imaging was performed on
DeltaVision OMX SR imaging system at the UCSF Nikon Imaging Center. We transfected U2OS cells with TMEM16K-V5, replated 6h later on high precision coverslips suitable for highperformance microscopy (Paul Marienfeld GmbH & Co, # 0117520, 1.5H, 12 mm ø), immunostained 24h later with mouse anti-V5, rabbit anti-Rab7 antibodies, and corresponding secondary antibodies, and mounted in Vectashield. Images are represented using pseudocolors suitable for color-blind palette.

BioID proximity biotinylation
To test whether the TMEM16K BioID-fusion proteins were enzymatically active and

Proteomic dataset analysis
TMEM16K proteome candidates list was generated from a minimum of 3 independent runs per condition. All proteins that had more than one peptide detected in control conditions were eliminated from further analysis. Next, only those proteins that were at least 3-fold enriched compared to control condition were considered potential interactors.

CI-MPR assay
CI-MPR assay was performed as previously reported with minor modifications 8,9 .

Transferrin internalization assay
For the pulse-chase experiment examining transferrin internalization, WT

EGF colocalization assay
After 16 hours of serum starvation, WT and TMEM16K KO cells were stimulated with 100 ng/ml of Alexa 647 conjugated EGF for 3 min at 37°C, and wash to remove EGF from the medium.
The cells were fixed at 10 min, 15 min, 40 min and 60 min after the initial exposure to Alexa 647-EGF, and immunostained with anti-Rab7 antibody. Images were acquired on the confocal microscope and colocalization between the Alexa647-EGF and Rab7 was analyzed in Fiji Software (ImageJ, NIH) using JaCOP plugin.

RUSH trafficking assay
The RUSH secretory assay was performed as previously published 10,11  Imaging System (LiCor).

Split-GFP assay
The split GFP system is based on GFP fragments containing β-strands 1-10 (GFP1-10) and β-strand 11 of GFP (GFP11) reconstituting complete β-barrel structure of GFP able to emit fluorescence when in sufficient proximity 13 . COS7 cells were transfected either with single constructs to verify their expression, localization and absence of signal in GFP channel, or double transfected to evaluate for the presence of the signal in the GFP channel. The imaging was done on Leica SP8 confocal microscope with same imaging parameters between conditions. Since TMEM16K forms a dimer 14,15 , we validated the split-GFP approach by expressing TMEM16K-GFP1-10 and GFP11-TMEM16K to reconstitute the split-GFP. We used proteins that are not considered TMEM16K interaction partners based on our proteomics, but are known to participate in similar processes/compartments (OSBPL8, OSBPL9, VPS35) as negative controls. Images are represented using pseudocolors suitable for color-blind palette.

Statistical analysis
We used Student's two-tailed t-test or single factor ANOVA with post-test Bonferronicorrected t-test. We used box plot to graphically visualize data with all box-plot elements defined; the box includes the first quartile and the third quartile, with the central line representing the median. Whiskers represent the minimum and maximum values of data. X inside the box represents the mean of data. No statistical method was used to determine sample size in any of the experiments.