Syndecan-1 Mediates Sorting of Soluble Lipoprotein Lipase with Sphingomyelin-Rich Membrane in the Golgi Apparatus

Abstract

In the secretory pathway, budding of vesicular transport carriers from the trans-Golgi network (TGN) must coordinate specification of lipid composition with selection of secreted proteins. We elucidate a mechanism of soluble protein cargo sorting into secretory vesicles with a sphingomyelin-rich membrane; the integral membrane proteoglycan Syndecan-1 (SDC1) acts as a sorting receptor, capturing the soluble enzyme lipoprotein lipase (LPL) during export from the TGN. Sorting of LPL requires bivalent interactions between LPL and SDC1-linked heparan sulfate chains and between LPL and the Golgi membrane. Physical features of the SDC1 transmembrane domain, rather than a specific sequence, confer targeting of SDC1 and bound LPL into the sphingomyelin secretion pathway. This study establishes that physicochemical properties of a protein transmembrane domain that drive lateral heterogeneity of the plasma membrane also operate at the TGN to confer sorting of an integral membrane protein and its ligand within the biosynthetic secretory pathway.

Keywords: Golgi apparatus; HSPGs; Syndecan-1; lipoprotein lipase; secretion; sphingomyelin.

Figure 1.. LPL exocytosis in vesicles of the SMS pathway requires sorting determinants in both domains.

(A) Time-lapse gallery of LPL, EQ-SM, and EQ-sol exocytosis. Galleries show example exocytic events of pHluorin-LPL and EQ-SM-mKate2 or EQ-sol-mKate2 captured by TIRFM. The corresponding graphs show total fluorescence intensity for each channel of the region of interest in each frame over time. (B) Full-length LPL, and not individual domains of LPL, is co-secreted with EQ-SM compared to EQ-sol. The mean proportions of exocytic events observed in at least 2 independent TIRFM experiments (± SD), where pHluorin-LPL, pHluorin-LPL*, pHluorin-lipase, or pHluorin-PLAT containing vesicles also contained EQ-SM-mKate2 or EQ-sol-mKate2 are indicated (251 events/13 cells for LPL+EQ-SM, 141 events/10 cells for LPL+EQ-sol, 249 events/13 cells for LPL*+EQ-SM, 246 events/13 cells for LPL*+EQ-sol, 219 events/15 cells for lipase+EQ-SM, 161 events/10 cells for lipase+EQ-sol, 554 events/17 cells for PLAT+EQ-SM, 357 events/12 cells for PLAT+EQ-sol). LPL* indicates a catalysis-deficient mutant form (S132A), and “lipase” and “PLAT” refer to LPL constructs containing residues 28–340 and 328–476, respectively. **** indicates p< 0.0001, n.s. indicates not significant (p>0.05). (C) Model of LPL domains. LPL is composed of two domains; lipase domain is yellow and PLAT domain is blue. The positions of four aromatic residues in the unstructured loop that were mutated (Y387A, W390A, W393A, W394A) in LPL-AR mutant protein are indicated with red circles. The heparin binding (HB) residues in clusters HB1 (K403, R405, D407), HB2 (K296, R297, K300), and HB3 (R279, K280, R282) are shown as magenta sticks. The structure was derived from PDB: 6E7K (Birrane, et al., 2019).

Figure 2.. Interaction of the LPL PLAT domain with membrane is required for sorting into the SMS pathway.

(A) LPL PLAT domain binds synthetic liposomes. Recombinant PLAT, PLAT-AR, and EQ-SM protein were incubated with liposomes containing 20% SM or PC (and 20% cholesterol), and liposomes were collected by centrifugation. Bound pellet (P) and unbound supernatant (S) fractions were visualized by Coomassie blue staining and quantified. (B) Quantification of liposome binding experiment. The proportion of bound protein out of the total is plotted as a mean value (± SD) for three independent experiments. (C) Aromatic residues in the PLAT domain are required for LPL co-secretion with EQ-SM. TIRFM-based exocytosis experiments were used to score the proportion of secretory vesicles containing pHluorin-LPL-AR and EQ-SM-mKate2 or EQ-sol-mKate2, without and with expression of GPIHBP1-Flag (means ± SD in at least 2 independent experiments; 185 events/10 cells for pH-LPL-AR+EQ-SM, 152 events/11 cells for pH-LPL-AR+EQ-sol, 179 events/12 cells for pH-LPL-AR+EQ-SM+GPIHBP1-Flag, 140 events/10 cells for pH-LPL-AR+EQ-sol+GPIHBP1-Flag).

Figure 3.. Interaction with heparin sulfate proteoglycans retains LPL on the cell surface and is required for LPL secretion via the SMS pathway.

(A) LPL localization in HeLa cells. Cells were transfected with GFP-LPL and visualized by TIRFM 16h after transfection. After secretion, LPL remains associated with the cell surface in untreated cells. Treatment of cells with 5 U/mL heparin for 5 minutes eliminates LPL cell surface localization. Micrograph shows one z-slice. Scale bars represent 10 μm. (B) Production of HSPGs is required for co-secretion of LPL and EQ-SM. TIRFM was used to score the proportion of pHluorin-LPL containing vesicles that also contained EQ-SM-mKate2 or EQ-sol-mKate2 in cells pretreated with DMSO or 2.5 mM xyloside for 48h. The means ± SD in at least 2 independent experiments are shown (222 events/12 cells for pH-LPL+EQ-SM+DMSO, 166 events/11 cells for pH-LPL+EQ-sol+DMSO, 160 events/11 cells for pH-LPL+EQ-SM+xyloside, 130 events/10 cells for pH-LPL+EQ-sol+xyloside). The diagrams below illustrate the effect of xyloside treatment on HSPG biosynthesis. (C) Mutation of heparin binding residues reduces LPL co-secretion with EQ-SM. The heparin binding residues in cluster 1 were mutated to Alanines and this construct, pHluorin-LPL-HB1, was used in TIRFM where co-secretion with EQ-SM-mKate2 and EQ-sol-mKate2 was scored. The means ± SD in at least 2 independent experiments are indicated (256 events/14 cells for LPL-HB1+EQ-SM, 190 events/11 cells for LPL-HB1+EQ-sol).

Figure 4:. Identification of Syndecan-1 as an LPL sorting receptor.

(A) Syndecan-1 is uniquely required for co-secretion of LPL and EQ-SM. Cells were transfected with a non-targeting siRNA or with siRNAs targeting Syndecan-1, Syndecan-2, Syndecan-3, or Syndecan-4 for 72h before TIRFM exocytosis experiments. Exocytic cargo loads were evaluated and the means ± SD in at least 2 independent experiments are shown (208 events/12 cells for pH-LPL+EQ-SM+si-C, 222 events/12 cells for pH-LPL+EQ-sol+si-C, 172 events/11 cells for pH-LPL+EQ-SM+si-SDC1, 160 events/10 cells for pH-LPL+EQ-sol+si-SDC1, 149 events/11 cells for pH-LPL+EQ-SM+si-SDC2, 196 events/12 cells for pH-LPL+EQ-sol+si-SDC2, 123 events/10 cells for pH-LPL+EQ-SM+si-SDC3, 140 events/10 cells for pH-LPL+EQ-sol+si-SDC3, 176 events/11 cells for pH-LPL+EQ-SM+si-SDC4, 154 events/12 cells for pH-LPL+EQ-sol+si-SDC4). (B) LPL and SDC1 copurify. Cells were transfected with plasmids encoding GFP-LPL and SDC1-Flag for 24h. Cell lysates were prepared and incubated with Flag beads for 1h. A representative Western blot shows input and immunipurified (IP) fractions for cells co-expressing GFP-LPL and SDC1-Flag, cells expressing only GFP-LPL, in vitro mixing of cells independently expressing GFP-LPL or SDC1-Flag. (C) LPL and SDC1 are co-secreted in the same vesicle population. TIRFM was used to score the co-secretion of pHluorin-LPL and pHluorin-hGH with RUSH-Ruby-SDC1. Cargo loads were scored 15–60 minutes after release of RUSH-Ruby-SDC1 by biotin addition. The means ± SD in at least 2 independent experiments are shown (157 events/11 cells for pH-LPL+RUSH-Ruby-SDC1, 192 events/11 cells for pH-hGH+RUSH-Ruby-SDC1). *** indicates p< 0.001

Figure 5:. The TMD of SDC1 drives secretion via the SMS pathway.

(A) Schematic of Syndecan-1. The core protein is shown in blue, with the TMD highlighted in purple. The amino acid sequences of SDC1’s TMD and TMD_L are shown. Small side chain residues are shown in purple and a putative palmitoylation site is shown in red. (B) Example micrographs of cells expressing RUSH-pHluorin-SDC1 or RUSH-pHluorin-SDC1-TMD_L. Both proteins predominantly localize to the perinuclear ER before release and show a prominent cell surface localization 30 minutes after the addition of biotin. Scale bars represent 10 μm. (C) The TMD_L sequence does not support co-secretion with EQ-SM. TIRFM scoring the co-secretion of RUSH-pHluorin-SDC1 and RUSH-pHluorin-SDC1-TMD_L with EQ-SM-mKate2 and EQ-sol-mKate2. The means ± SD in at least 2 independent experiments are shown (264 events/11 cells for pH-SDC1+EQ-SM, 281 events/11 cells for pH-SDC1+EQ-sol, 260 events/11 cells for pH-SDC1-TMD_L+EQ-SM, 270 events/11 cells for pH-SDC1-TMD_L+EQ-sol). ** indicates p< 0.01. (D) The Ala-Leu transmembrane domain sequence confers secretion by the SMS pathway. Cells were transfected with plasmids to express paired combinations of RUSH-TMD_A8L, RUSH-TMD_L, EQ-SM, and EQ-sol. TIRFM was used to score the mean proportion of co-secretion ± SD in at least 2 independent experiments (192 events/11 cells for TMD_A8L+EQ-SM, 291 events/12 cells for TMD_A8L+EQ-sol, 192 events/10 cells for TMD_L+EQ-SM, 330 events/15 cells for TMD_L+EQ-sol, 219 events/13 cells for pH-TMD_A8L+Ruby-TMD_L, and 245 events/12 cells for Ruby-TMD_A8L + pH-TMD_L).

Figure 6:. SDC1 TMD and heparan chains are required for LPL secretion via the SMS pathway.

(A) Schematic diagram depicting wild type and mutant SDC1 proteins. Note the larger volume of the TMD in SDC1-TMD_L and the absence of the three heparan sulfate chains at the N-terminus of SDC1-HS. (B) Micrographs comparing the localization of wild type SCD1 and mutants. Cells were transfected with SDC1-Flag, SDC1-TMD_L-Flag, or SDC1-HS-Flag and antisera to Flag was used to detect each protein by immunofluorescence microscopy. Scale bars indicate 10 μm. (C) SDC1-TMD_L and SDC1-HS plasmids fail to rescue LPL sorting in cells depleted of endogenous SDC1. Cells were transfected with siRNAs targeting Syndecan-1 and with plasmid expressing si-RNA-resistant SDC1, SDC1-TMD_L, or SDC1-HS 72h before TIRFM sorting experiments. Exocytic cargo loads were evaluated and the means ± SD in at least 2 independent experiments are shown (185 events/11 cells for pH-LPL+EQ-SM+si-SDC1+SDC1, 133 events/11 cells for pH-LPL+EQ-sol+si-SDC1+SDC1, 198 events/11 cells for pH-LPL+EQ-SM+si-SDC1+SDC1-TMD_L, 139 events/11 cells for pH-LPL+EQ-sol+si-SDC1+SDC1-TMD_L, 191 events/12 cells for pH-LPL+EQ-SM+si-SDC1+SDC1-HS, and 180 events/12 cells for pH-LPL+EQ-sol+si-SDC1+SDC1-HS). Sorting data for pH-LPL+EQ-SM/EQ-sol in SDC1-depleted cells is reproduced from Figure 4A. (D) GFP-LPL co-immunopurification with SDC1 requires heparan sulfate chains. Cells were transfected with GFP-LPL and SDC1-Flag, SDC1-TMD_L-Flag, or SDC1-HS-Flag (shown in panel A) for 24h and SDC1-Flag was immunopurified as in Figure 4B. A representative Western blot shows input and IP.

Figure 7:. Model of SDC1 mediated LPL secretion.

Aromatic residues (red) in the LPL PLAT domain localize LPL to the membrane, where it is captured by SDC1 via binding of LPL heparin binding residues to Syndecan-1’s heparan sulfate chains. The SDC1 TMD drives the concentration of Syndecan-1 and bound LPL in sphingomyelin-rich membrane, enriching these cargos in vesicles of the SMS pathway as they bud from the TGN.