AP-1 Recruits SMAP-1/SMAPs to the trans-Golgi Network to Promote Sorting in Polarized Epithelia

Coordinated AP-1 and clathrin coat assembly mediate secretory sorting on the trans-Golgi network (TGN) during conventional secretion. Here we found that SMAP-1/SMAPs deficiency caused the apical protein ERM-1 to accumulate on the basolateral side of the TGN. In contrast, the basolateral protein SLCF-1 appeared abnormally on the apical membrane. SMAP-1 colocalized with AP-1 on the TGN. The integrity of AP-1 is required for the subcellular presence of SMAP-1. Moreover, we found that the loss of SMAP-1 reduced clathrin-positive structures in the cytosol, suggesting that SMAP-1 has a regulatory role in clathrin assembly on the TGN. Functional experiments showed that overexpressing clathrin effectively alleviated exocytic defects due to the lack of SMAP-1, corroborating the role of SMAP-1 in promoting the assembly of clathrin on the TGN. Together, our results suggested that the AP-1 complex regulates the TGN localization of SMAP-1, promoting clathrin assembly to ensure polarized conventional secretion in C. elegans intestinal epithelia.


INTRODUCTION
In the conventional secretion pathway, cargo proteins traverse ER-Golgi and reach the plasma membrane via transport vesicles (Mellman and Warren, 2000;Rabouille, 2017;Dimou and Nickel, 2018). There are apical and basolateral membrane domains in epithelial cells, which leads to additional complexity of cargo sorting (Yeaman et al., 1999;Ang et al., 2003;Sato et al., 2007;Nakajo et al., 2016). Accumulating evidence indicated that the trans-Golgi network (TGN) functions as a sorting organelle during secretion in epithelial cells (Mellman and Warren, 2000;Gravotta et al., 2007;Thuenauer et al., 2014). Apical and basolateral proteins must be separated in TGN before their inclusion into separate routes. To ensure polarized secretion, delicate sorting machinery is employed to package the cargo proteins into specific vesicles and then deliver them to various downstream destinations. TGN missorting can lead to inappropriate targeting of cargo proteins and cell polarity defects (Guo et al., 2014).
Here, we introduced SMAP-1 (stromal membrane-associated protein-1) as a polarized secretion regulator in C. elegans intestinal epithelia. SMAP-1 overlapped well with TGN markers, AP-1, and clathrin. Notably, the presence of the AP-1 complex was essential for SMAP-1 localization. Furthermore, we found that SMAP-1 deficiency led to a loss of TGN localization of clathrin. Overexpression of clathrin instead of AP-1 component effectively alleviated secretion defects. In summary, our results suggested that AP-1 governs the TGN In smap-1(ycxEx1639) mutants, ERM-1-GFP and NHX-2-GFP accumulated on the basolateral side, while SLCF-1-GFP and LET-413-GFP appeared on the apical membrane. White asterisks indicate intestinal lumen. Error bars are 95% CIs (n 18 each). Asterisks designate the significant differences in the Mann-Whitney test (***p < 0.001). Scale bars, 10 μm. localization of SMAP-1, and SMAP-1 acts to facilitate clathrin assembly during polarized secretion.

Loss of SMAP-1 Disturbs the Polarized Secretion in the Intestine
To better understand the regulatory mechanisms of polarized secretion in epithelia, we deployed apically localized ERM-1-GFP (a membrane-cytoskeleton linker) and basolateral SLCF-1-GFP (a monocarboxylate transporter) as cargos for a genome-wide RNAi screen. We found that loss of W09D10.1 led to defective secretion in the C. elegans intestine. W09D10.1 encodes the sole C. elegans homolog of mammalian SMAPs (Funaki et al., 2013;Sato et al., 1998;Tanabe et al., 2005), which we referred to as SMAP-1. Sequence alignment indicated that the N-terminal region (aa 15-119) of SMAP-1 shares 68% identity with SMAP2, while their C-terminal regions lack significant homology. SMAP-1 contains an N-terminal Arf GAP domain (aa 20-128) ( Figure 1B). Unlike the C-terminus of SMAP2, which harbors a clathrin-interacting domain and a CALM-interacting domain (Natsume et al., 2006), the C-terminal part of SMAP-1 has not been characterized yet.
To verify the role of ARF-1.2 in SMAP-mediated cargo sorting, we examined the localization of ERM-1 and SLCF-1. Notably, the distribution of ERM-1-GFP and SLCF-1-GFP was affected in arf-1.2(RNAi) animals (Supplementary Figure S3C-C9). However, ARF-1.2 knockdown failed to alleviate the distributional defects of ERM-1 and SLCF-1 in SMAP-1deficient cells (Supplementary Figure S3C-C9). Conversely, overexpression of SMAP-1 (R60A)-mCherry rescued the distributional defects of ERM-1 and SLCF-1 (Supplementary Figure S3D-D9). It is noteworthy that Arf1 GTPase has been shown to recruit AP-1 to facilitate the formation of the clathrin coat on the trans-Golgi network (TGN) (Ren et al., 2013;Beacham et al., 2019). Altogether, our results suggested that in addition to acting as a GAP of ARF-1.2 during polarized cargo sorting, SMAP-1 plays an additional role in facilitating sorting in C. elegans intestinal epithelia.
ARF-1.2 is required for the retrograde transport from Golgi to the endoplasmic reticulum (ER) (Arakel et al., 2019). To this end, we set to examine the localization of COPB-1 (COP-I complex subunit beta 1), which is expressed in the intestine (Hunt-Newbury et al., 2007;Ackema et al., 2014). As expected, COPB-1 predominantly colocalized with Golgi marker mCherry-P4M (Supplementary Figure S4B-B9). In the absence of SMAP-1, the level of colocalization between COPB-1-GFP and MC-P4M was decreased significantly (Supplementary Figure S4B-B9). Similarly, the Golgi localization of COPG-1 (COP-I complex subunit gamma 1) was reduced (Supplementary Figure S4A-A9). These results indicated that the increase in ARF-1.2 activity induced by SMAP-1 deficiency affected the Golgi recruitment of COP-I. Alternatively, SMAP-1 could directly participate in the assembly of COP-I coatomer.
Additionally, Arf1 has been reported to promote the Golgi association of gamma ear-containing Arf-binding proteins (GGAs) (Doray et al., 2002;D'Souza-Schorey and Chavrier, 2006), which cooperates with clathrin in cargo sorting. In C. elegans, APT-9 is the homolog of human GGA1. To determine whether SMAP-1 affects the localization of APT-9/GGA1, we assessed the distribution of APT-9-GFP. In the absence of SMAP-1, although APT-9-GFP accumulated in the cytosol, the localization of APT-9-GFP in mCherry-P4M-labeled Golgi apparatus was significantly reduced (Supplementary Figure  S4C-D9). These results suggested that SMAP-1 also functions as a negative regulator of APT-9/GGA1 in C. elegans intestinal cells. However, the increase in ARF-1.2 activity does not seem to affect the Golgi association of APT-9/GGA1 directly.

Localization of SMAP-1 in the trans-Golgi Network Requires AP-1
Previous studies showed that the AP-1 complex mediates clathrin assembly and acts synergistically with clathrin to regulate sorting on the TGN (Robinson and Bonifacino, 2001). The punctate structures labeled by CHC-1 (clathrin heavy chain) were consistently reduced in the absence of AP-1 subunits ( Figure  3A-A9). In addition, depleting AP-1 subunits or clathrin caused ERM-1 to accumulate around the basolateral membrane and resulted in the presence of SLCF-1 in the apical membrane of intestinal cells (Supplementary Figure S6A-A9). Remarkably, most SMAP-1-GFP-labeled structures also disappeared upon loss of AP-1 subunits ( Figure 3A-A9). It is noteworthy that the C. elegans genome encodes an additional AP-1 μ1 subunit UNC-101 (Shim et al., 2000), which has been implicated in the polarized sorting of KVS-4 in DA9 motor neurons (Zhou et al., 2016). Conversely, loss of UNC-101 did not disturb the distributional of CHC-GFP (Supplementary Figure S6C-C9), supporting the distinct, tissue-specific functions of APM-1 and UNC-101 (Shim et al., 2000).
Frontiers in Cell and Developmental Biology | www.frontiersin.org November 2021 | Volume 9 | Article 774401 6 (σ1), we performed GST pull-down assays. We found that SMAP-1 was bound to APG-1, while there was no significant interaction between SMAP-1 and APM-1, APB-1, and APS-1 ( Figures  3D-G). In contrast, we did not observe the interaction between SMAP-1 and clathrin heavy chain (CHC-1) ( Figure 3H). Together, these data suggested that the integrity of the AP-1 complex is required for SMAP-1 localization in TGN and that the clathrin assembly event likely occurs downstream of SMAP-1.

Loss of SMAP-1 Leads to Reduced Clathrin Coat Assembly in the TGN
Thus far, our analysis revealed that clathrin assembly is likely to occur downstream of SMAP-1. To further clarify the effect of FIGURE 5 | Loss of SMAP-1 leads to a decrease in TGN-located clathrin. (A-A9) Loss of SMAP-1 led to a decrease in the colocalization between CHC-1 and P4M. Pearson's correlation coefficients are calculated, error bars are 95% CIs (n 12 animals). p-value: Mann-Whitney test. ***p < 0.001. (B-B9) The colocalization between APM-1 and P4M was not affected by the depletion of SMAP-1. Pearson's correlation coefficients are calculated, error bars are 95% CIs (n 12 animals). p-value: Mann-Whitney test. ns: no significance. (C-C9) In the absence of SMAP-1, CHC-1 failed to overlap with APG-1 in punctate structures. Pearson's correlation coefficients are calculated, error bars are 95% CIs (n 12 animals). p-value: Mann-Whitney test. ***p < 0.001. Scale bars, 10 μm.
Frontiers in Cell and Developmental Biology | www.frontiersin.org November 2021 | Volume 9 | Article 774401 SMAP-1 on clathrin localization, we compared clathrin with the PI(4)P marker GFP-P4M in the absence of SMAP-1. Remarkably, loss of SMAP-1 reduced the overlap between residual CHC-1-GFP and mCherry-P4M ( Figure 5A-A9), validating that SMAP-1/SMAP2 regulates the occurrence of clathrin in the Golgi apparatus. However, the Golgi localization of APM-1 was not affected upon loss of SMAP-1 ( Figure 5B-B9). We subsequently examined the level of colocalization between CHC-1-GFP and mCherry-APG-1. In the absence of SMAP-1, we found that the remaining CHC-1-labeled structure no longer colocalized with APG-1 ( Figure 5C-C9). In addition to biosynthetic sorting, the clathrin coat is known to mediate the formation of endocytic clathrin-coated vesicles (Chen and Schmid, 2020;Moulay et al., 2020). Also, clathrin has been reported to function as a component of the retrograde transport machinery on the surface of the endosome (Saint-Pol et al., 2004;Shi et al., 2009). Therefore, the punctate structures distinct from the P4M-or APG-1-positive puncta are likely clathrin-coated vesicles and sorting endosomes ( Figure 5A-A9, C-C9). Taken together, our results indicated that SMAP-1 acts as an indispensable regulator that directs TGN clathrin coat assembly downstream of the AP-1 complex.

DISCUSSION
Here, we identified SMAP-1/SMAPs as a polarized secretion regulator in the C. elegans intestine. SMAP-1 colocalizes with AP-1 and clathrin in the TGN. The integrity of the AP-1 complex is required for SMAP-1 positioning, and SMAP-1 acts to sustain clathrin assembly to ensure AP-1/clathrin-dependent cargos sorting (Supplementary Figure S7). Studies in mammals indicated that SMAP1 functions as an Arf6GAP to regulate clathrin-dependent endocytosis via binding directly to clathrin (Tanabe et al., 2005). Additionally, SMAP2 was implicated in endosome-to-Golgi retrograde transport (Natsume et al., 2006). A recent study showed that SMAP2 facilitates clathrin assembly protein (CALM) mediated formation of clathrin-coated carriers on the TGN, promoting acrosome formation (Funaki et al., 2013). Together, these results suggested that SMAPs are clathrin assembly regulators in the TGN, and this efficacy could be due to its ArfGAP activity. In the current study, we found that SMAP-1 regulates polarized sorting, and this function seems independent of ARF-1.2, supporting the role of SMAPs as secretion regulators. Furthermore, our study highlighted the diversity of SMAPs functionality and corroborated the significance of AP-1/clathrin coat assembly in polarized sorting.
Previous studies have shown that SMAPs interact with clathrin and CALM, modulating clathrin-coated vesicle formation on the TGN (Tanabe et al., 2005;Natsume et al., 2006;Funaki et al., 2013). However, the mechanism controlling the localization of SMAPs is still not well understood. Here, by using a well-established in vivo membrane trafficking investigation model (Chen et al., 2018;Chen et al., 2019;Gao et al., 2020;Zhang et al., 2020;Yan et al., 2021), we showed that the integrity of AP-1 adaptor is necessary for the TGN positioning of SMAP-1. Although we did not specifically identify which AP-1 subunit governs the TGN localization of SMAP-1, our results suggested that in addition to CALM, SMAP-1 underlies an additional clathrin assembly mechanism, enriching the understanding of AP-1/clathrin coat assembly. It is reasonable to speculate that a similar mechanism might be involved in the budding of clathrin-coated vesicles during endocytosis. Further analyses are required to dissect the details of this biological process.

C. elegans Strains
Genetic crosses of C. elegans were performed by standard methods (Brenner, 1974). A list of strains was provided in Supplemental Materials. RNAi-mediated gene expression interference was implemented by the feeding protocol (Timmons and Fire, 1998). RNAi constructs were from the Ahringer library (Kamath and Ahringer, 2003). For chc-1, apb-1, apg-1, apm-1, and aps-1 RNAi experiments, L2-L3 stage larvae were cultured for 48-60h and scored as adults.

Microscopy and Image Analysis
Live animals were mounted on 2% agarose pads (100 mM levamisole). Fluorescence images were obtained with a Nikon C2 laser scanning confocal microscope (Nikon, Tokyo, Japan) equipped with a 100×N.A. 1.2 oil-immersion objective. Images were collected with NIS-Elements AR 4.40.00 software. Z-series of optical sections were acquired using 0.8-1 μm step size. Fluorescence data were evaluated with Metamorph software version 7.10.3.279 (Universal Imaging, West Chester, PA). The "Integrated Morphometry Analysis" component was utilized to assess the fluorescence intensity (total intensity), puncta number (structure count), and fluorescence area (total area) within unit regions. For each genotype, a total of 6 animals were analyzed in three unit regions of each intestine defined by a 100 × 100 (pixl 2 ) box located randomly (n 18 each). In this case, "total area" is a comprehensive parameter indicating the number and size of the fluorescent structures. Colocalization images were analyzed by Fiji (Image J) software (Schindelin et al., 2012). Pearson's correlation coefficients were calculated with 6 animals for all genotypes.

Statistical Analysis
Prism software version 8.02 (GraphPad Software, La Jolla, CA) was deployed to perform statistical analyses.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

AUTHOR CONTRIBUTIONS
SW and AS designed the study; SW and LY performed the experiments; WZ, ZC, CH, HL, and YY contributed the reagents; SW and AS analyzed the data; SW and AS wrote the paper with inputs and final approval from all authors.

ACKNOWLEDGMENTS
We are grateful to Xin Zhang, Xin Fu, and Zhenrong Yang for their assistance. Error bars are 95% CIs (n 18 each, 9 animals of each genotype were sampled in whole-cell regions of two intestinal cells). Asterisks indicate the significant differences in the Mann-Whitney test (***p < 0.001, *p < 0.05). (B-B9) The membrane-to-cytosol ratio (P/S) of ARF-1.2-GFP increased in smap-1(RNAi) animals. Membrane structures were separated from the cytosol of worm lysates by ultracentrifugation. ARF-1.2-GFP in the supernatants and pellets were analyzed by western blotting using an anti-GFP antibody. The loading control was blotted by the anti-Tubulin antibody. The SEMs from three independent experiments are shown, asterisk indicates the significant differences in the one-tailed Student's ttest (***p < 0.001). (C-C9) Confocal images of the intestinal cells expressing GFPtagged ERM-1 and SLCF-1. Asterisks in the panels indicate intestinal lumen. Error bars are 95% CIs (n 18 each, 9 animals of each genotype were sampled in wholecell regions of two intestinal cells). Asterisks indicate the significant differences in the Mann-Whitney test (***p < 0.001, ns: no significance). (D-D9) Confocal images of the intestinal cells expressing GFP-tagged ERM-1 and SLCF-1. In smap-1(ycxEx1639) mutants, overexpression of SMAP-1(R60A)-mCherry that has lost GAP activity rescued the distribution defects of ERM-1-GFP and SLCF-1-GFP. Asterisks in the panels indicate intestinal lumen. Error bars are 95% CIs (n 18 each, 9 animals of each genotype were sampled in whole-cell regions of two intestinal cells). Asterisks indicate the significant differences in the Mann-Whitney test (***p < 0.001, ns: no significance). Scale bars, 10 μm. November 2021 | Volume 9 | Article 774401 9 (n 12 animals). P-value: Mann-Whitney test. ***p < 0.001. (B-B9) Confocal images showing colocalization between COPB-1 and Golgi marker P4M in the intestinal cells. Arrowheads indicate structures labeled by both GFP and mCherry. Pearson's correlation coefficients for GFP and mCherry signals are calculated, error bars are 95% CIs (n 12 animals). P-value: Mann-Whitney test. ***p < 0.001. (C-C9) Confocal images showing colocalization between APT-9 and Golgi marker P4M in the intestinal cells. Arrowheads indicate structures labeled by both GFP and mCherry. Pearson's correlation coefficients for GFP and mCherry signals are calculated, error bars are 95% CIs (n 12 animals). P-value: Mann-Whitney test. ***p < 0.001. (D-D9) Confocal images showing APT-9-GFP in the intestinal cells. Error bars are 95% CIs (n 18 each, 9 animals of each genotype were sampled in whole-cell regions of two intestinal cells). Asterisks indicate the significant differences in the Mann-Whitney test (***p < 0.001). Scale bars, 10 μm.
Supplementary Figure S6 | (A-A9) Confocal images of the intestinal cells expressing GFP-tagged ERM-1 and SLCF-1. Asterisks in the panels indicate intestinal lumen. Error bars are 95% CIs (n 18 each, 9 animals of each genotype were sampled in whole-cell regions of two intestinal cells). Asterisks indicate the significant differences in a one-way ANOVA followed by a post-hoc test (Dunn's Multiple Comparison Test) for multiple comparisons (***p < 0.001). (B-B9) Confocal image showing colocalization between GFP-APM-1 (μ1 subunit) and SMAP-1-mCherry in the intestinal cells. Arrowheads indicate structures labeled by both GFP and mCherry. Pearson's correlation coefficients for GFP and mCherry signals are calculated, error bars are 95% CIs (n 12 animals). (C-C9) Confocal images showing CHC-1-GFP in the intestinal cells. Error bars are 95% CIs (n 18 each, 9 animals of each genotype were sampled in whole-cell regions of two intestinal cells). Asterisks indicate the significant differences in the Mann-Whitney test (ns: no significance). Scale bars, 10 μm.
Supplementary Figure S7 | In the C. elegans intestine, ARF-1.2 regulates clathrin coat assembly via downstream AP-1 and SMAP-1, promoting TGN sorting. In addition, SMAP-1 acts as a GAP to regulate ARF-1.2 activity in a negative feedback manner.