Tunnels for Protein Export from the Endoplasmic Reticulum.

The functions of coat protein complex II (COPII) coats in cargo packaging and the creation of vesicles at the endoplasmic reticulum are conserved in eukaryotic protein secretion. Standard COPII vesicles, however, cannot handle the secretion of metazoan-specific cargoes such as procollagens, apolipoproteins, and mucins. Metazoans have thus evolved modules centered on proteins like TANGO1 (transport and Golgi organization 1) to engage COPII coats and early secretory pathway membranes to engineer a novel mode of cargo export at the endoplasmic reticulum.

Auto-regulation of Secretory Flux by Sensing and Responding to the Folded Cargo Protein Load in the Endoplasmic Reticulum.

Maintaining the optimal performance of cell processes and organelles is the task of auto-regulatory systems. Here we describe an auto-regulatory device that helps to maintain homeostasis of the endoplasmic reticulum (ER) by adjusting the secretory flux to the cargo load. The cargo-recruiting subunit of the coatomer protein II (COPII) coat, Sec24, doubles as a sensor of folded cargo and, upon cargo binding, acts as a guanine nucleotide exchange factor to activate the signaling protein Gα12 at the ER exit sites (ERESs). This step, in turn, activates a complex signaling network that activates and coordinates the ER export machinery and attenuates proteins synthesis, thus preventing large fluctuations of folded and potentially active cargo that could be harmful to the cell or the organism. We call this mechanism AREX (autoregulation of ER export) and expect that its identification will aid our understanding of human physiology and diseases that develop from secretory dysfunction.

Segregation of nascent GPCRs in the ER-to-Golgi transport by CCHCR1 via direct interaction.

G protein-coupled receptors (GPCRs) constitute the largest superfamily of cell surface signaling proteins that share a common structural topology. When compared with agonist-induced internalization, how GPCRs are sorted and delivered to functional destinations after synthesis in the endoplasmic reticulum (ER) is much less well understood. Here, we demonstrate that depletion of coiled-coil α-helical rod protein 1 (CCHCR1) by siRNA and CRISPR-Cas9 significantly inhibits surface expression and signaling of α2A-adrenergic receptor (α2A-AR; also known as ADRA2A), without affecting α2B-AR. Further studies show that CCHCR1 depletion specifically impedes α2A-AR export from the ER to the Golgi, but not from the Golgi to the surface. We also demonstrate that CCHCR1 selectively interacts with α2A-AR. The interaction is mediated through multiple domains of both proteins and is ionic in nature. Moreover, mutating CCHCR1-binding motifs significantly attenuates ER-to-Golgi export, surface expression and signaling of α2A-AR. Collectively, these data reveal a novel function for CCHCR1 in intracellular protein trafficking, indicate that closely related GPCRs can be sorted into distinct ER-to-Golgi transport routes by CCHCR1 via direct interaction, and provide important insights into segregation and anterograde delivery of nascent GPCR members.

Selective inhibition of protein secretion by abrogating receptor-coat interactions during ER export.

Protein secretion is an essential process that drives cell growth, movement, and communication. Protein traffic within the secretory pathway occurs via transport intermediates that bud from one compartment and fuse with a downstream compartment to deliver their contents. Here, we explore the possibility that protein secretion can be selectively inhibited by perturbing protein-protein interactions that drive capture into transport vesicles. Human proprotein convertase subtilisin/kexin type 9 (PCSK9) is a determinant of cholesterol metabolism whose secretion is mediated by a specific cargo adaptor protein, SEC24A. We map a series of protein-protein interactions between PCSK9, its endoplasmic reticulum (ER) export receptor SURF4, and SEC24A that mediate secretion of PCSK9. We show that the interaction between SURF4 and SEC24A can be inhibited by 4-phenylbutyrate (4-PBA), a small molecule that occludes a cargo-binding domain of SEC24. This inhibition reduces secretion of PCSK9 and additional SURF4 clients that we identify by mass spectrometry, leaving other secreted cargoes unaffected. We propose that selective small-molecule inhibition of cargo recognition by SEC24 is a potential therapeutic intervention for atherosclerosis and other diseases that are modulated by secreted proteins.

A systematic review of Sec24 cargo interactome.

Endoplasmic reticulum (ER)-to-Golgi trafficking is an essential and highly conserved cellular process. The coat protein complex-II (COPII) arm of the trafficking machinery incorporates a wide array of cargo proteins into vesicles through direct or indirect interactions with Sec24, the principal subunit of the COPII coat. Approximately one-third of all mammalian proteins rely on the COPII-mediated secretory pathway for membrane insertion or secretion. There are four mammalian Sec24 paralogs and three yeast Sec24 paralogs with emerging evidence of paralog-specific cargo interaction motifs. Furthermore, individual paralogs also differ in their affinity for a subset of sorting motifs present on cargo proteins. As with many aspects of protein trafficking, we lack a systematic and thorough understanding of the interaction of Sec24 with cargoes. This systematic review focuses on the current knowledge of cargo binding to both yeast and mammalian Sec24 paralogs and their ER export motifs. The analyses show that Sec24 paralog specificity of cargo (and cargo receptors) range from exclusive paralog dependence or preference to partial redundancy. We also discuss how the Sec24 secretion system is hijacked by viral (eg, VSV-G, Hepatitis B envelope protein) and bacterial (eg, the enteropathogenic Escherichia coli type III secretion system effector NleA/EspI) pathogens.

Export of polybasic motif-containing secretory proteins BMP8A and SFRP1 from the endoplasmic reticulum is regulated by surfeit locus protein 4.

In the conventional secretory pathway, cargo receptors play important roles in exporting newly synthesized secretory proteins from the endoplasmic reticulum (ER). We previously showed that a cargo receptor, surfeit locus protein 4 (SURF4), promotes ER export of a soluble signaling molecule, sonic hedgehog, via recognizing the polybasic residues within its Cardin-Weintraub motif. In addition to sonic hedgehog, we found 30 more secretory proteins containing the polybasic motif (K/R)(K/R)(K/R)XX(K/R)(K/R), but whether SURF4 plays a general role in mediating ER export of these secretory proteins is unclear. Here, we analyzed the trafficking of four of these secretory proteins: desert hedgehog, Indian hedgehog, bone morphogenetic protein 8A (BMP8A), and secreted frizzled-related protein 1 (SFRP1). We found that the polybasic motifs contained in these cargo proteins are important for their ER export. Further analyses indicated that the polybasic motifs of BMP8A and SFRP1 interact with the triacidic motif on the predicted first luminal domain of SURF4. These interactions with SURF4 are essential and sufficient for the ER-to-Golgi trafficking of BMP8A and SFRP1. Moreover, we demonstrated that SURF4 localizes at a subpopulation of ER exit sites to regulate the ER export of its clients. Taken together, these results suggest that SURF4 is recruited to specific ER exit sites and plays a general role in capturing polybasic motif-containing secretory cargo proteins through electrostatic interactions.

The Ubp3/Bre5 deubiquitylation complex modulates COPII vesicle formation.

The appropriate delivery of secretory proteins to the correct subcellular destination is an essential cellular process. In the endoplasmic reticulum (ER), secretory proteins are captured into COPII vesicles that generally exclude ER resident proteins and misfolded proteins. We previously characterized a collection of yeast mutants that fail to enforce this sorting stringency and improperly secrete the ER chaperone, Kar2 (Copic et al., Genetics 2009). Here, we used the emp24Δ mutant strain that secretes Kar2 to identify candidate proteins that might regulate ER export, reasoning that loss of regulatory proteins would restore sorting stringency. We find that loss of the deubiquitylation complex Ubp3/Bre5 reverses all of the known phenotypes of the emp24Δ mutant, and similarly reverses Kar2 secretion of many other ER retention mutants. Based on a combination of genetic interactions and live cell imaging, we conclude that Ubp3 and Bre5 modulate COPII coat assembly at ER exit sites. Therefore, we propose that Ubp3/Bre5 influences the rate of vesicle formation from the ER that in turn can impact ER quality control events.

Diacidic Motifs in the Carboxyl Terminus Are Required for ER Exit and Translocation to the Plasma Membrane of NKCC2.

Mutations in the apical Na-K-2Cl co-transporter, NKCC2, cause type I Bartter syndrome (BS1), a life-threatening kidney disease. We have previously demonstrated that the BS1 variant Y998X, which deprives NKCC2 from its highly conserved dileucine-like motifs, compromises co-transporter surface delivery through ER retention mechanisms. However, whether these hydrophobic motifs are sufficient for anterograde trafficking of NKCC2 remains to be determined. Interestingly, sequence analysis of NKCC2 C-terminus revealed the presence of consensus di-acidic (D/E-X-D/E) motifs, 949EEE951 and 1019DAELE1023, located upstream and downstream of BS1 mutation Y998X, respectively. Di-acidic codes are involved in ER export of proteins through interaction with COPII budding machinery. Importantly, whereas mutating 949EEE951 motif to 949AEA951 had no effect on NKCC2 processing, mutating 1019DAE1021 to 1019AAA1021 heavily impaired complex-glycosylation and cell surface expression of the cotransporter in HEK293 and OKP cells. Most importantly, triple mutation of D, E and E residues of 1019DAELE1023 to 1019AAALA1023 almost completely abolished NKCC2 complex-glycosylation, suggesting that this mutant failed to exit the ER. Cycloheximide chase analysis demonstrated that the absence of the terminally glycosylated form of 1019AAALA1023 was caused by defects in NKCC2 maturation. Accordingly, co-immunolocalization experiments revealed that 1019AAALA1023 was trapped in the ER. Finally, overexpression of a dominant negative mutant of Sar1-GTPase abolished NKCC2 maturation and cell surface expression, clearly indicating that NKCC2 export from the ER is COPII-dependent. Hence, our data indicate that in addition to the di-leucine like motifs, NKCC2 uses di-acidic exit codes for export from the ER through the COPII-dependent pathway. We propose that any naturally occurring mutation of NKCC2 interfering with this pathway could form the molecular basis of BS1.

The chaperone Chs7 forms a stable complex with Chs3 and promotes its activity at the cell surface.

The polytopic yeast protein Chs3 (chitin synthase III) relies on a dedicated membrane-localized chaperone, Chs7, for its folding and expression at the cell surface. In the absence of Chs7, Chs3 forms high molecular weight aggregates and is retained in the endoplasmic reticulum (ER). Chs7 was reported to be an ER resident protein, but its role in Chs3 folding and transport was not well characterized. Here, we show that Chs7 itself exits the ER and localizes with Chs3 at the bud neck and intracellular compartments. We identified mutations in the Chs7 C-terminal cytosolic domain that do not affect its chaperone function, but cause it to dissociate from Chs3 at a post-ER transport step. Mutations that prevent the continued association of Chs7 with Chs3 do not block delivery of Chs3 to the cell surface, but dramatically reduce its catalytic activity. This suggests that Chs7 engages in functionally distinct interactions with Chs3 to first promote its folding and ER exit, and subsequently to regulate its activity at the plasma membrane.

Tango1 coordinates the formation of endoplasmic reticulum/Golgi docking sites to mediate secretory granule formation.

Regulated secretion is a conserved process occurring across diverse cells and tissues. Current models suggest that the conserved cargo receptor Tango1 mediates the packaging of collagen into large coat protein complex II (COPII) vesicles that move from the endoplasmic reticulum (ER) to the Golgi apparatus. However, how Tango1 regulates the formation of COPII carriers and influences the secretion of other cargo remains unknown. Here, through high-resolution imaging of Tango1, COPII, Golgi, and secretory cargo (mucins) in Drosophila larval salivary glands, we found that Tango1 forms ring-like structures that mediate the formation of COPII rings rather than vesicles. These COPII rings act as docking sites for the cis-Golgi. Moreover, we observed nascent secretory mucins emerging from the Golgi side of these Tango1-COPII-Golgi complexes, suggesting that these structures represent functional docking sites/fusion points between the ER exit sites and the Golgi. Loss of Tango1 disrupted the formation of COPII rings, the association of COPII with the cis-Golgi, mucin O-glycosylation, and secretory granule biosynthesis. Additionally, we identified a Tango1 self-association domain that is essential for formation of this structure. Our results provide evidence that Tango1 organizes an interaction site where secretory cargo is efficiently transferred from the ER to Golgi and then to secretory vesicles. These findings may explain how the loss of Tango1 can influence Golgi/ER morphology and affect the secretion of diverse proteins across many tissues.

Signal motif-dependent ER export of the Qc-SNARE BET12 interacts with MEMB12 and affects PR1 trafficking in Arabidopsis.

Soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) are well-known for their role in controlling membrane fusion, the final, but crucial step, in vesicular transport in eukaryotes. SNARE proteins contribute to various biological processes including pathogen defense and channel activity regulation, as well as plant growth and development. Precise targeting of SNARE proteins to destined compartments is a prerequisite for their proper functioning. However, the underlying mechanism(s) for SNARE targeting in plants remains obscure. Here, we investigate the targeting mechanism of the Arabidopsis thaliana Qc-SNARE BET12, which is involved in protein trafficking in the early secretory pathway. Two distinct signal motifs that are required for efficient BET12 ER export were identified. Pulldown assays and in vivo imaging implicated that both the COPI and COPII pathways were required for BET12 targeting. Further studies using an ER-export-defective form of BET12 revealed that the Golgi-localized Qb-SNARE MEMB12, a negative regulator of pathogenesis-related protein 1 (PR1; At2g14610) secretion, was its interacting partner. Ectopic expression of BET12 caused no inhibition in the general ER-Golgi anterograde transport but caused intracellular accumulation of PR1, suggesting that BET12 has a regulatory role in PR1 trafficking in A. thaliana.

EB1- and EB2-dependent anterograde trafficking of TRPM4 regulates focal adhesion turnover and cell invasion.

Transient receptor potential melastatin 4 (TRPM4) is a Ca2+-activated nonselective cationic channel involved in a wide variety of physiologic and pathophysiological processes. Bioinformatics analyses of the primary sequence of TRPM4 allowed us to identify a putative motif for interaction with end-binding (EB) proteins, which are microtubule plus-end tracking proteins. Here, we provide novel data suggesting that TRPM4 interacts with EB proteins. We show that mutations of the putative EB binding motif abolish the TRPM4-EB interaction, leading to a reduced expression of the mature population of the plasma membrane channel and instead display an endoplasmic reticulum-associated distribution. Furthermore, we demonstrate that EB1 and EB2 proteins are required for TRPM4 trafficking and functional activity. Finally, we demonstrated that the expression of a soluble fragment containing the EB binding motif of TRPM4 reduces the plasma membrane expression of the channel and affects TRPM4-dependent focal adhesion disassembly and cell invasion processes.-Blanco, C., Morales, D., Mogollones, I., Vergara-Jaque, A., Vargas, C., Álvarez, A., Riquelme, D., Leiva-Salcedo, E., González, W., Morales, D., Maureira, D., Aldunate, I., Cáceres, M., Varela, D., Cerda, O. EB1- and EB2-dependent anterograde trafficking of TRPM4 regulates focal adhesion turnover and cell invasion.

The fifth subunit in α3ß4 nicotinic receptor is more than an accessory subunit.

The α3ß4 subtype is the predominant neuronal nicotinic acetylcholine receptor present in the sensory and autonomic ganglia and in a subpopulation of brain neurons. This subtype can form pentameric receptors with either 2 or 3 ß4 subunits that have different pharmacologic and functional properties. To further investigate the role of the fifth subunit, we coexpressed a dimeric construct coding for a single polypeptide containing the ß4 and α3 subunit sequences, with different monomeric subunits. With this strategy, which allowed the formation of single populations of receptors with unique stoichiometry, we demonstrated with immunofluorescence and biochemical and functional assays that only the receptors with 3 ß4 subunits are efficiently expressed at the plasma membrane. Moreover, the LFM export motif of ß4 subunit in the fifth position exerts a unique function in the regulation of the intracellular trafficking of the receptors, their exposure at the cell surface, and consequently, their function, whereas the same export motif present in the ß4 subunits forming the acetylcholine binding site is dispensable.-Crespi, A., Plutino, S., Sciaccaluga, M., Righi, M., Borgese, N., Fucile, S., Gotti, C., Colombo, S. F. The fifth subunit in α3ß4 nicotinic receptor is more than an accessory subunit.

Selective export of autotaxin from the endoplasmic reticulum.

Autotaxin (ATX) or ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2) is a secretory glycoprotein and functions as the key enzyme for lysophosphatidic acid generation. The mechanism of ATX protein trafficking is largely unknown. Here, we demonstrated that p23, a member of the p24 protein family, was the protein-sorting receptor required for endoplasmic reticulum (ER) export of ATX. A di-phenylalanine (Phe-838/Phe-839) motif in the human ATX C-terminal region was identified as a transport signal essential for the ATX-p23 interaction. Knockdown of individual Sec24 isoforms by siRNA revealed that ER export of ATX was impaired only if Sec24C was down-regulated. These results suggest that ATX is selectively exported from the ER through a p23, Sec24C-dependent pathway. In addition, it was found that AKT signaling played a role in ATX secretion regulation to facilitate ATX ER export by enhancing the nuclear factor of activated T cell-mediated p23 expression. Furthermore, the di-hydrophobic amino acid motifs (FY) also existed in the C-terminal regions of human ENPP1 and ENPP3. Such a p23, Sec24C-dependent selective ER export mechanism is conserved among these ENPP family members.

Inhibition of cargo export at ER exit sites and the trans-Golgi network by the secretion inhibitor FLI-06.

Export out of the endoplasmic reticulum (ER) involves the Sar1 and COPII machinery acting at ER exit sites (ERES). Whether and how cargo proteins are recruited upstream of Sar1 and COPII is unclear. Two models are conceivable, a recruitment model where cargo is actively transported through a transport factor and handed over to the Sar1 and COPII machinery in ERES, and a capture model, where cargo freely diffuses into ERES where it is captured by the Sar1 and COPII machinery. Using the novel secretion inhibitor FLI-06, we show that recruitment of the cargo VSVG to ERES is an active process upstream of Sar1 and COPII. Applying FLI-06 before concentration of VSVG in ERES completely abolishes its recruitment. In contrast, applying FLI-06 after VSVG concentration in ERES does not lead to dispersal of the concentrated VSVG, arguing that it inhibits recruitment to ERES as opposed to capture in ERES. FLI-06 also inhibits export out of the trans-Golgi network (TGN), suggesting that similar mechanisms might orchestrate cargo selection and concentration at the ER and TGN. FLI-06 does not inhibit autophagosome biogenesis and the ER-peroxisomal transport route, suggesting that these rely on different mechanisms.

SLC6 Transporter Folding Diseases and Pharmacochaperoning.

The human genome encodes 19 genes of the solute carrier 6 (SLC6) family; non-synonymous changes in the coding sequence give rise to mutated transporters, which are misfolded and thus cause diseases in the affected individuals. Prominent examples include mutations in the transporters for dopamine (DAT, SLC6A3), for creatine (CT1, SLC6A8), and for glycine (GlyT2, SLC6A5), which result in infantile dystonia, mental retardation, and hyperekplexia, respectively. Thus, there is an obvious unmet medical need to identify compounds, which can remedy the folding deficit. The pharmacological correction of folding defects was originally explored in mutants of the serotonin transporter (SERT, SLC6A4), which were created to study the COPII-dependent export from the endoplasmic reticulum. This led to the serendipitous discovery of the pharmacochaperoning action of ibogaine. Ibogaine and its metabolite noribogaine also rescue several disease-relevant mutants of DAT. Because the pharmacology of DAT and SERT is exceptionally rich, it is not surprising that additional compounds have been identified, which rescue folding-deficient mutants. These compounds are not only of interest for restoring DAT function in the affected children. They are also likely to serve as useful tools to interrogate the folding trajectory of the transporter. This is likely to initiate a virtuous cycle: if the principles underlying folding of SLC6 transporters are understood, the design of pharmacochaperones ought to be facilitated.

Unique COPII component AtSar1a/AtSec23a pair is required for the distinct function of protein ER export in Arabidopsis thaliana.

Secretory proteins traffic from endoplasmic reticulum (ER) to Golgi via the coat protein complex II (COPII) vesicle, which consists of five cytosolic components (Sar1, Sec23-24, and Sec13-31). In eukaryotes, COPII transport has diversified due to gene duplication, creating multiple COPII paralogs. Evidence has accumulated, revealing the functional heterogeneity of COPII paralogs in protein ER export. Sar1B, the small GTPase of COPII machinery, seems to be specialized for large cargo secretion in mammals. Arabidopsis contains five Sar1 and seven Sec23 homologs, and AtSar1a was previously shown to exhibit different effects on α-amylase secretion. However, mechanisms underlying the functional diversity of Sar1 paralogs remain unclear in higher organisms. Here, we show that the Arabidopsis Sar1 homolog AtSar1a exhibits distinct localization in plant cells. Transgenic Arabidopsis plants expressing dominant-negative AtSar1a exhibit distinct effects on ER cargo export. Mutagenesis analysis identified a single amino acid, Cys84, as being responsible for the functional diversity of AtSar1a. Structure homology modeling and interaction studies revealed that Cys84 is crucial for the specific interaction of AtSar1a with AtSec23a, a distinct Arabidopsis Sec23 homolog. Structure modeling and coimmunoprecipitation further identified a corresponding amino acid, Cys484, on AtSec23a as being essential for the specific pair formation. At the cellular level, the Cys484 mutation affects the distinct function of AtSec23a on vacuolar cargo trafficking. Additionally, dominant-negative AtSar1a affects the ER export of the transcription factor bZIP28 under ER stress. We have demonstrated a unique plant pair of COPII machinery function in ER export and the mechanism underlying the functional diversity of COPII paralogs in eukaryotes.

A Conserved Di-Basic Motif of Drosophila Crumbs Contributes to Efficient ER Export.

The Drosophila type I transmembrane protein Crumbs is an apical determinant required for the maintenance of apico-basal epithelial cell polarity. The level of Crumbs at the plasma membrane is crucial, but how it is regulated is poorly understood. In a genetic screen for regulators of Crumbs protein trafficking we identified Sar1, the core component of the coat protein complex II transport vesicles. sar1 mutant embryos show a reduced plasma membrane localization of Crumbs, a defect similar to that observed in haunted and ghost mutant embryos, which lack Sec23 and Sec24CD, respectively. By pulse-chase assays in Drosophila Schneider cells and analysis of protein transport kinetics based on Endoglycosidase H resistance we identified an RNKR motif in Crumbs, which contributes to efficient ER export. The motif identified fits the highly conserved di-basic RxKR motif and mediates interaction with Sar1. The RNKR motif is also required for plasma membrane delivery of transgene-encoded Crumbs in epithelial cells of Drosophila embryos. Our data are the first to show that a di-basic motif acts as a signal for ER exit of a type I plasma membrane protein in a metazoan organism.

Release from endoplasmic reticulum matrix proteins controls cell surface transport of MHC class I molecules.

The anterograde transport of secretory proteins from the endoplasmic reticulum (ER) to the plasma membrane is a multi-step process. Secretory proteins differ greatly in their transport rates to the cell surface, but the contribution of each individual step to this difference is poorly understood. Transport rates may be determined by protein folding, chaperone association in the ER, access to ER exit sites (ERES) and retrieval from the ER-Golgi intermediate compartment or the cis-Golgi to the ER. We have used a combination of folding and trafficking assays to identify the differential step in the cell surface transport of two natural allotypes of the murine major histocompatibility complex (MHC) class I peptide receptor, H-2D(b) and H-2K(b) . We find that a novel pre-ER exit process that acts on the folded lumenal part of MHC class I molecules and that drastically limits their access to ERES accounts for the transport difference of the two allotypes. Our observations support a model in which the cell surface transport of MHC class I molecules and other type I transmembrane proteins is governed by the affinity of all their folding and maturation states to the proteins of the ER matrix.

Diverse subcellular localizations of the insect CMP-sialic acid synthetases.

The occurrence and biological importance of sialic acid (Sia) and its metabolic enzymes in insects have been studied using Drosophila melanogaster. The most prominent feature of D. melanogaster CMP-Sia synthetase (DmCSS) is its Golgi-localization, contrasted with nuclear localization of vertebrate CSSs. However, it remains unclear if the Golgi-localization is common to other insect CSSs and why it happens. To answer these questions, Aedes aegypti (mosquito) CSS (AaCSS) and Tribolium castaneum (beetle) CSS (TcCSS) were cloned and characterized for their activity and subcellular localization. Our new findings show: (1) AaCSS and TcCSS share a common overall structure with DmCSS in terms of evolutionarily conserved motifs and the absence of the C-terminal domain typical to vertebrate CSSs; (2) when expressed in mammalian and insect cells, AaCSS and TcCSS showed in vivo and in vitro CSS activities, similar to DmCSS. In contrast, when expressed in bacteria, they lacked CSS activity because the N-terminal hydrophobic region appeared to induce protein aggregation; (3) when expressed in Drosophila S2 cells, AaCSS and TcCSS were predominantly localized in the ER, but not in the Golgi. Surprisingly, DmCSS was mainly secreted into the culture medium, although partially detected in Golgi. Consistent with these results, the N-terminal hydrophobic regions of AaCSS and TcCSS functioned as a signal peptide to render them soluble in the ER, while the N-terminus of DmCSS functioned as a membrane-spanning region of type II transmembrane proteins whose cytosolic KLK sequence functioned as an ER export signal. Accordingly, the differential subcellular localization of insect CSSs are distinctively more diverse than previously recognized.