A SURF4-to-proteoglycan relay mechanism that mediates the sorting and secretion of a tagged variant of sonic hedgehog

Sonic Hedgehog (Shh) is a key signaling molecule that plays important roles in various developmental processes in mammals. Although the signal transduction pathway activated by Shh is well understood, the regulation of its secretion remains unclear. Newly synthesized Shh is imported into the endoplasmic reticulum (ER), where it undergoes a series of posttranslational modifications to produce the mature lipid-modified amino-terminal fragment. Here, we have analyzed the molecular mechanisms that mediate secretion of the N-terminal fragment of Shh (ShhN). We found that the Cardin–Weintraub (CW) motif in Shh is necessary and sufficient for ER-to-Golgi transport of ShhN. Mechanistic analyses revealed that a cargo receptor, Surfeit locus protein 4 (SURF4), interacts directly with the CW motif of ShhN to regulate packaging of ShhN into COPII vesicles. ShhN and SURF4 interact with each other at the ER and separate from each other after entering the Golgi. The CW motif is known to interact with proteoglycans (PGs) that are predominantly synthesized at the Golgi. Interestingly, we found that PGs compete with SURF4 to bind ShhN and that inhibiting synthesis of PGs causes defects in export of ShhN from the trans Golgi network (TGN). SURF4 and PG maturation are also important for intracellular traffic of full length Shh in mammalian cells. Our study suggests a SURF4-to-PG relay mechanism that mediates the sorting and secretion of Shh, providing insight into the biosynthetic trafficking of Shh.

The Hedgehog (Hh) signaling pathway plays an important role in various developmental processes in metazoans (1, 2). Mutations of key components that regulate Hh signaling are associated with many human diseases (3). Hh was first found in the Drosophila larval epidermis. It mediates larval segment development and adult appendage patterning (4). In mammals, there are three Hh-family members, Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh). Ihh regulates the proliferation and differentiation of chondrocytes (5). Dhh functions in gonads, regulating testis organogenesis, spermatogenesis (6, 7), and follicle development in the ovary (8). Shh functions more extensively than the other two Hh members: it regulates embryonic patterning (4), specification of cell types in the nervous system (9), axon guidance (10), cell differentiation, and organ development (11).Hh is synthesized as a full-length precursor Hh (HhFL). After entering the endoplasmic reticulum (ER), HhFL is autocleaved into two parts: an N-terminal Hedge domain (HhN) and a C-terminal Hog domain (HhC) (1). HhC is degraded through ER-associated degradation (12). HhN undergoes lipid modifications, in which a cholesterol molecule is covalently linked to the C terminus and a palmitoyl group is linked to the N terminus (13–15). Lipid-modified HhN subsequently exits the ER and is delivered via the secretory pathway to the plasma membrane. Once at the plasma membrane, Hh is released into the extracellular matrix and ultimately recognized by its receptors on the plasma membrane of target cells to induce downstream signal transduction.Although significant progress has been achieved in understanding the Hh signaling pathway in target cells, the molecular mechanisms that mediate secretion of newly synthesized Shh proteins from the producing cells are still unclear. The ER is the first station where newly synthesized proteins enter the secretory pathway. In this compartment, cargo proteins are generally recognized by the coat protein complex II (COPII) to be packaged into vesicles and exported from the ER. Soluble cargo proteins in the ER lumen cannot directly engage the COPII coat but instead are captured into vesicles by transmembrane cargo receptors. One mammalian cargo receptor, ERGIC53, is a mannose-specific lectin that recognizes N-linked glycoproteins in the ER lumen (16, 17). The p24 family of proteins function as cargo receptors to regulate ER export of glycosylphosphatidylinositol (GPI)-anchored proteins (18). Mammalian orthologs of yeast ER vesicle (Erv) proteins have also been thought to function as cargo receptors (16). Surfeit locus protein 4 (SURF4), the mammalian ortholog of Erv29p, regulates ER export of soluble proteins, including lipoproteins and proprotein convertase subtilisin/kexin type 9 (PCSK9) (19–21). SURF4 recognizes amino-terminal tripeptide motifs of soluble cargo proteins and participates in ER exit site (ERES) organization (19, 22). The cargo receptors that mediate sorting of Shh in the secretory pathway remain unknown.Here, we examined trafficking of the N-terminal fragment of Shh without the cholesterol modification (referred to as ShhN). We utilized the Retention Using Selective Hook (RUSH) assay (23) to analyze the kinetics of trafficking of ShhN along the secretory pathway. We reconstituted the packaging of ShhN into transport vesicles in vitro and utilized this assay to quantitatively measure packaging efficiency. Our study reveals cellular factors and underlying mechanisms that mediate the sorting and secretion of Shh, providing insight into the biosynthetic trafficking of Shh.