Sorting to synaptic-like microvesicles from early and late endosomes requires overlapping but not identical targeting signals

In PC12 neuroendocrine cells, synaptic-like microvesicles (SLMV) are thought to be formed by two pathways. One pathway sorts the proteins to SLMV directly from the plasma membrane (or a specialized domain thereof) in an adaptor protein complex 2-dependent, brefeldin A (BFA)-insensitive manner. Another pathway operates via an endosomal intermediate, involves adaptor protein complex 3, and is BFA sensitive. We have previously shown that when expressed in PC12 cells, HRP-P-selectin chimeras are directed to SLMV mostly via the endosomal, BFA-sensitive route. We have now found that two endosomal intermediates are involved in targeting of HRP-P-selectin chimeras to SLMV. The first intermediate is the early, transferrin-positive, epidermal growth factor-positive endosome, from which exit to SLMV is controlled by the targeting determinants YGVF and KCPL, located within the cytoplasmic domain of P-selectin. The second intermediate is the late, transferrin-negative, epidermal growth factor-positive late endosome, from where HRP-P-selectin chimeras are sorted to SLMV in a YGVF- and DPSP-dependent manner. Both sorting steps, early endosomes to SLMV and late endosomes to SLMV, are affected by BFA. In addition, analysis of double mutants with alanine substitutions of KCPL and YGVF or KCPL and DPSP indicated that chimeras pass sequentially through these intermediates en route both to lysosomes and to SLMV. We conclude that a third site of formation for SLMV, the late endosomes, exists in PC12 cells.     

Targeting of P-selectin to two regulated secretory organelles in PC12 cells

Targeting of P-selectin to the regulated secretory organelles (RSOs) of phaeochromocytoma PC12 cells has been investigated. By expressing from cDNA a chimera composed of HRP and P-selectin, and then following HRP activity through subcellular fractionation, we have discovered that P- selectin contains signals that target HRP to the synaptic-like microvesicles (SLMV) as well as the dense-core granules (DCGs) of these cells. Mutagenesis of the chimera followed by transient expression in PC12 cells shows that at least two different sequences within the carboxy-terminal cytoplasmic tail of P-selectin are necessary, but that neither is sufficient for trafficking to the SLMV. One of these sequences is centred on the 10 amino acids of the membrane-proximal C1 exon that is also implicated in lysosomal targeting. The other sequence needed for trafficking to the SLMV includes the last four amino acids of the protein. The same series of mutations have a different effect on DCG targeting, showing that traffic to the two different RSOs depends on different features within the cytoplasmic domain of P-selectin.     

P-selectin targeting to secretory lysosomes of Rbl-2H3 cells.

The biogenesis of secretory lysosomes, which combine characteristics of both lysosomes and secretory granules, is currently of high interest. In particular, it is not clear whether delivery of membrane proteins to the secretory lysosome requires lysosomal, secretory granule, or some novel targeting determinants. Heterologous expression of P-selectin has established that this membrane protein contains targeting signals for both secretory granules and lysosomes. P-selectin is therefore an ideal probe with which to determine the signals required for targeting to secretory lysosomes. We have exploited subcellular fractionation and immunofluorescence microscopy to monitor targeting of transiently expressed wild-type and mutant horseradish peroxidase (HRP)-P-selectin chimeras to secretory lysosomes of Rbl-2H3 cells. The exposure of the HRP chimeras to intracellular proteolysis was also determined as a third monitor of secretory lysosome targeting. Our data show that HRP-P-selectin accumulates in secretory lysosomes of Rbl-2H3 cells using those cytoplasmic sequences previously found to be sufficient for targeting to conventional lysosomes. This work highlights the similar sorting signals used for targeting of membrane proteins to conventional lysosomes and secretory lysosomes.     

Synaptic vesicles form by budding from tubular extensions of sorting endosomes in PC12 cells

The putative role of sorting early endosomes (EEs) in synaptic-like microvesicle (SLMV) formation in the neuroendocrine PC12 cell line was investigated by quantitative immunoelectron microscopy. By BSA-gold internalization kinetics, four distinct endosomal subcompartments were distinguished: primary endocytic vesicles, EEs, late endosomes, and lysosomes. As in other cells, EEs consisted of vacuolar and tubulovesicular subdomains. The SLMV marker proteins synaptophysin and vesicle-associated membrane protein 2 (VAMP-2) localized to both the EE vacuoles and associated tubulovesicles. Quantitative analysis showed that the transferrin receptor and SLMV proteins colocalized to a significantly higher degree in primary endocytic vesicles then in EE-associated tubulovesicles. By incubating PC12 cells expressing T antigen-tagged VAMP (VAMP-TAg) with antibodies against the luminal TAg, the recycling pathway of SLMV proteins was directly visualized. At 15 degrees C, internalized VAMP-TAg accumulated in the vacuolar domain of EEs. Upon rewarming to 37 degrees C, the labeling shifted to the tubular part of EEs and to newly formed SLMVs. Our data delineate a pathway in which SLMV proteins together with transferrin receptor are delivered to EEs, where they are sorted into SLMVs and recycling vesicles, respectively.     

Di-leucine signals mediate targeting of tyrosinase and synaptotagmin to synaptic-like microvesicles within PC12 cells

One pathway in forming synaptic-like microvesicles (SLMV) involves direct budding from the plasma membrane, requires adaptor protein 2 (AP2) and is brefeldin A (BFA) resistant. A second route leads from the plasma membrane to an endosomal intermediate from which SLMV bud in a BFA-sensitive, AP3-dependent manner. Because AP3 has been shown to bind to a di-leucine targeting signal in vitro, we have investigated whether this major class of targeting signals is capable of directing protein traffic to SLMV in vivo. We have found that a di-leucine signal within the cytoplasmic tail of human tyrosinase is responsible for the majority of the targeting of HRP-tyrosinase chimeras to SLMV in PC12 cells. Furthermore, we have discovered that a Met-Leu di-hydrophobic motif within the extreme C terminus of synaptotagmin I supports 20% of the SLMV targeting of a CD4-synaptotagmin chimera. All of the traffic to the SLMV mediated by either di-Leu or Met-Leu is BFA sensitive, strongly suggesting a role for AP3 and possibly for an endosomal intermediate in this process. The differential reduction in SLMV targeting for HRP-tyrosinase and CD4-synaptotagmin chimeras by di-alanine substitutions or BFA treatment implies that different proteins use the two routes to the SLMV to differing extents.     

Secretagogue-triggered Transfer of Membrane Proteins from Neuroendocrine Secretory Granules to Synaptic-like Microvesicles

The membrane proteins of all regulated secretory organelles (RSOs) recycle after exocytosis. However, the recycling of those membrane proteins that are targeted to both dense core granules (DCGs) and synaptic-like microvesicles (SLMVs) has not been addressed. Since neuroendocrine cells contain both RSOs, and the recycling routes that lead to either organelle overlap, transfer between the two pools of membrane proteins could occur during recycling. We have previously demonstrated that a chimeric protein containing the cytosolic and transmembrane domains of P-selectin coupled to horseradish peroxidase is targeted to both the DCG and the SLMV in PC12 cells. Using this chimera, we have characterized secretagogue-induced traffic in PC12 cells. After stimulation, this chimeric protein traffics from DCGs to the cell surface, internalizes into transferrin receptor (TFnR)-positive endosomes and thence to a population of secretagogue-responsive SLMVs. We therefore find a secretagogue-dependent rise in levels of HRP within SLMVs. In addition, the levels within SLMVs of the endogenous membrane protein, synaptotagmin, as well as a green fluorescent protein-tagged version of vesicle-associated membrane protein (VAMP)/synaptobrevin, also show a secretagogue-dependent increase.     

AP-3 adaptor functions in targeting P-selectin to secretory granules in endothelial cells

P-selectin, a cell adhesion protein participating in the early stages of inflammation, contains multiple sorting signals that regulate its cell surface expression. Targeting to secretory granules regulates delivery of P-selectin to the cell surface. Internalization followed by sorting from early to late endosomes mediates rapid removal of P-selectin from the surface. We show here that the P-selectin cytoplasmic domain bound AP-2 and AP-3 adaptor complexes in vitro . The amino acid substitution L768A, which abolishes endosomal sorting and impairs granule targeting of P-selectin, reduced binding of AP-3 adaptors but not AP-2 adaptors. Turnover of P-selectin was 2.4-fold faster than turnover of transferrin receptor in AP-3-deficient mocha fibroblasts, similar to turnover of these two proteins in AP-3-competent cells, demonstrating that AP-3 function is not required for endosomal sorting. However, sorting P-selectin to secretory granules was defective in endothelial cells from AP-3-deficient pearl mice, demonstrating a role for AP-3 adaptors in granule assembly in endothelial cells. P-selectin sorting to platelet α-granules was normal in pearl mice, consistent with earlier evidence that granule targeting of P-selectin is mechanistically distinct in endothelial cells and platelets. These observations establish that AP-3 adaptor functions in assembly of conventional secretory granules, in addition to lysosomes and the 'lysosome-like' secretory granules of platelets and melanocytes.     

Cellugyrin and synaptogyrin facilitate targeting of synaptophysin to a ubiquitous synaptic vesicle-sized compartment in PC12 cells

Cellugyrin represents a ubiquitously expressed four-transmembrane domain protein that is closely related to synaptic vesicle protein synaptogyrin and, more remotely, to synaptophysin. We report here that, in PC12 cells, cellugyrin is localized in synaptic-like microvesicles (SLMVs), along with synaptogyrin and synaptophysin. Upon overexpression of synaptophysin in PC12 cells, it is localized in rapidly sedimenting membranes and practically is not delivered to the SLMVs. On the contrary, the efficiency of the SLMV targeting of exogenously expressed cellugyrin and synaptogyrin is high. Moreover, expression of cellugyrin (or synaptogyrin) in PC12 cells dramatically and specifically increases SLMV targeting of endogenous synaptophysin. Finally, we utilized the SLMV purification scheme on a series of non-neuroendocrine cell types including the mouse fibroblast cell line 3T3-L1, the Chinese hamster ovary cell line CHO-K1, and the monkey kidney epithelial cell line COS7 and found that a cellugyrin-positive microvesicular compartment was present in all cell types tested. We suggest that synaptic vesicles have evolved from cellugyrin-positive ubiquitous microvesicles and that neuroendocrine SLMVs represent a step along that pathway of evolution.     

The calcium-sensing protein synaptotagmin 7 is expressed on different endosomal compartments in endocrine,neuroendocrine cells or neurons but not on large dense core vesicles

Synaptotagmin (syt) isoforms function as calcium sensor in post-Golgi transport although the precise transport step and compartment(s) concerned are still not fully resolved. As syt7 has been proposed to operate in lysosomal exocytosis and in exocytosis of large dense core vesicles (LDCVs), we have addressed the distribution of endogenous syt7 in insulin-secreting cells. These cells express different syt7 isoforms comparable to neurons. According to subcellular fractionation and quantitative confocal immunocytochemistry, syt7 is not found on LDCVs or on synaptic-like microvesicles but colocalizes with Rab7 on endosomes and to structures near to or at the plasma membrane. Similarly, endogenous syt7 was absent from LDCVs in pheochromocytoma PC12 cells. In contrast, syt7 localised to lysosomes in both, PC12 cells and hippocampal neurons. In conclusion, endogenous syt7 shows a wider distribution than previously reported but does not qualify as vesicular calcium sensor in SLMV or LDCV exocytosis according to its localisation.     

The cytoplasmic domain of P-selectin contains a sorting determinant that mediates rapid degradation in lysosomes

P-selectin is a cell adhesion molecule required transiently on the surface of activated platelets and endothelial cells as a receptor for leukocytes. It is stored in secretory granules in platelets, endothelial cells, and transfected neuroendocrine cells and is rapidly delivered to the plasma membrane upon exocytosis of the secretory granules. It is then rapidly internalized in endothelial cells and transfected cells. We find that in transfected neuroendocrine PC12 cells, the fraction of P-selectin that is not targeted to secretory granules is rapidly degraded. In transfected CHO fibroblasts, which lack secretory granules, P-selectin was degraded with a half time of 2.3 h in plated cells, while low density lipoprotein receptor (LDL-R) had a half life of 9 h. In cells cultured in ammonium chloride to inhibit lysosomal proteinases, P-selectin was protected from degradation and rapidly accumulated in vesicles enriched in lgp-B, a resident lysosomal membrane protein. The cytoplasmic domain of P- selectin was sufficient to confer rapid turnover on LDL-R. Deletion of 10 amino acids from the cytoplasmic domain of P-selectin extended the half life to 9.5 h and abrogated rapid lysosomal targeting in the presence of ammonium chloride, implicating this sequence as a necessary element of a novel lysosomal targeting signal. The rate limiting step in degradation occurred after internalization from the cell surface, indicating that sorting of P-selectin away from efficiently recycled proteins occurs in endosomes. We propose that this sorting event represents a constitutive equivalent of receptor down regulation, and may function to regulate the expression of P-selectin at the surface of activated endothelial cells.     

The intracellular trafficking of opioid receptors directed by carboxyl tail and a di-leucine motif in Neuro2A cells

The mu- and delta-opioid receptors (MOR and DOR) differ significantly in their intracellular trafficking. MORs recycle back to the cell surface upon agonist treatment, whereas most internalized DORs are targeted to lysosomes for degradation. By exchanging the carboxyl tail domains of MOR and DOR and expressing the receptor chimeras in mouse neuroblastoma Neuro2A cells, it could be demonstrated that the carboxyl tail domain is not the sole determinant in directing the intracellular trafficking in these Neuro2A cells. Deletion of the dileucine motif (Leu245-Leu246) within the third intracellular loop of DOR or the mutation of Leu245 to Met slowed the lysosomal targeting of these delta-opioid receptors. Meanwhile the mutation of Met264 to Leu increased the rate of agonist-induced receptor internalization and the lysosomal targeting of the wild type and the delta-opioid receptor carboxyl tail chimera of the mu-opioid receptor. These studies suggest interplay between a di-leucine motif and the carboxyl tail in the lysosomal targeting of the receptor.     

Divergent fates of P- and E-selectins after their expression on the plasma membrane.

P-selectin and E-selectin are related adhesion receptors for monocytes and neutrophils that are expressed by stimulated endothelial cells. P-selectin is stored in Weibel-Palade bodies, and it reaches the plasma membrane after exocytosis of these granules. E-selectin is not stored, and its synthesis is induced by cytokines. We studied the fate of the two proteins after their surface expression by following the intracellular routing of internalized antibodies to the selectins. By immunofluorescent staining, P-selectin antibody was first seen in endosomes, then in the Golgi region, and finally in Weibel-Palade bodies. In contrast, the E-selectin antibody was detected only in endosomes and lysosomes. Subcellular fractionation of cells after 4 h chase confirmed the localization of P-selectin antibody in storage granules and of the E-selectin antibody in lysosomes. In AtT-20 cells, a mouse pituitary cell line, transfected with P- or E-selectin, only P-selectin was delivered to the endogenous adrenocorticotrophic hormone storage granules after endocytosis. Deletion of the cytoplasmic domain abolished internalization. In summary, after a brief surface exposure, internalized E-selectin is degraded in the lysosomes, whereas P-selectin returns to the storage granules from where it can be reused.     

The pathway and targeting signal for delivery of the integral membrane glycoprotein LEP100 to lysosomes

A complete set of chimeras was made between the lysosomal membrane glycoprotein LEP100 and the plasma membrane-directed vesicular stomatitis virus G protein, combining a glycosylated lumenal or ectodomain, a single transmembrane domain, and a cytosolic carboxyl-terminal domain. These chimeras, the parent molecules, and a truncated form of LEP100 lacking the transmembrane and cytosolic domains were expressed in mouse L cells. Only LEP100 and chimeras that included the cytosolic 11 amino acid carboxyl terminus of LEP100 were targeted to lysosomes. The other chimeras accumulated in the plasma membrane, and truncated LEP100 was secreted. Chimeras that included the extracellular domain of vesicular stomatitis G protein and the carboxyl terminus of LEP100 were targeted to lysosomes and very rapidly degraded. Therefore, in chimera-expressing cells, virtually all the chimeric molecules were newly synthesized and still in the biosynthesis and lysosomal targeting pathways. The behavior of one of these chimeras was studied in detail. After its processing in the Golgi apparatus, the chimera entered the plasma membrane/endosome compartment and rapidly cycled between the plasma membrane and endosomes before going to lysosomes. In pulse-expression experiments, a large population of chimeric molecules was observed to appear transiently in the plasma membrane by immunofluorescence microscopy. Soon after protein synthesis was inhibited, this surface population disappeared. When lysosomal proteolysis was inhibited, chimeric molecules accumulated in lysosomes. These data suggest that the plasma membrane/early endosome compartment is on the pathway to the lysosomal membrane. This explains why mutations that block endocytosis result in the accumulation of lysosomal membrane proteins in the plasma membrane.     

Weibel-Palade body membrane proteins exhibit differential trafficking after exocytosis in endothelial cells

Weibel-Palade bodies, the secretory granules of endothelial cells, possess two different membrane proteins. However, P-selectin is seen only in Weibel-Palade bodies in HUVECs, whereas CD63 is also seen in late endosomes/lysosomes. Since P-selectin is targeted to lysosomes in heterologous expression studies, we have determined whether a lysosomal targeting signal also operates within HUVECs. We have also examined the trafficking of CD63 to its two different intracellular locations. By following antibodies bound at the plasma membrane during stimulation, we have discovered that while half of the P-selectin recycles to the WPBs, 50% is rapidly delivered to a lamp-1-positive compartment. Thus, the lysosomal targeting signal of this protein also operates in HUVECs. CD63 is found constitutively at the cell surface of HUVECs and most of it is delivered to the late endosomes/lysosomes after internalisation. However, stimulation causes both a rise in the CD63 plasma membrane level and in the amount that recycles to the WPBs. Our data strongly suggest that the CD63 that originates in the WPB preferentially recycles to the granule rather than being delivered to the late endosome/lysosome, and that there are, therefore, two separate pools of this protein within HUVECs. Our findings indicate that although P-selectin and CD63 are both targeted to the same compartments from the PM, the kinetics and the ratio of their targeting to Weibel-Palade bodies versus lysosomes are very different.     

Cytoplasmic Signals Mediate Apical Early Endosomal Targeting of Endotubin in MDCK Cells

Endotubin is an integral membrane protein that targets into apical endosomes in polarized epithelial cells. Although the role of cytoplasmic targeting signals as mediators of basolateral targeting and endocytosis is well established, it has been suggested that apical targeting requires either N-glycosylation of the ectoplasmic domains or partitioning of macromolecules into glycolipid-rich rafts. However, we have previously shown that the cytoplasmic portion of endotubin possesses signals that are necessary for its proper sorting into the apical early endosomes. To further define the targeting signals involved in this apically directed event, as well as to determine if the cytoplasmic domain was sufficient to mediate apical endosomal targeting, we generated a panel of endotubin and Tac-antigen chimeras and expressed them in Madin–Darby canine kidney cells. We show that both the apically targeting wild-type endotubin and a basolaterally targeted cytoplasmic domain mutant do not associate with rafts and are TX-100 soluble. The cytoplasmic tail of endotubin is sufficient for apical endosomal targeting, as chimeras with the endotubin cytoplasmic domain and Tac transmembrane and extracellular domains are efficiently targeted to the apical endosomal compartment. Furthermore, we show that overexpression of these chimeras results in their missorting to the basolateral membrane, indicating that the apical sorting process is a saturable event. These results show that cells contain machinery in both the biosynthetic and endosomal compartments that recognize cytoplasmic apical sorting signals.     

Different Steady State Subcellular Distributions of the Three Splice Variants of Lysosome-associated Membrane Protein LAMP-2 Are Determined Largely by the COOH-terminal Amino Acid Residue

The extensively glycosylated lysosome-associated membrane proteins (LAMP)-2a, b, and c are derived from a single gene by alternative splicing that produces proteins with differences in the transmembrane and cytosolic domains. The lysosomal targeting signals reside in the cytosolic domain of these proteins. LAMPs are not restricted to lysosomes but can also be found in endosomes and at the cell surface. We investigated the subcellular distribution of chimeras comprised of the lumenal domain of avian LAMP-1 and the alternatively spliced domains of avian LAMP-2. Chimeras with the LAMP-2c cytosolic domain showed predominantly lysosomal distribution, while higher levels of chimeras with the LAMP-2a or b cytosolic domain were present at the cell surface. The increase in cell surface expression was due to differences in the recognition of the targeting signals and not saturation of intracellular trafficking machinery. Site-directed mutagenesis defined the COOH-terminal residue of the cytosolic tail as critical in governing the distributions of LAMP-2a, b, and c between intracellular compartments and the cell surface.     

Endosomal Sorting of Amyloid Precursor Protein-P-Selectin Chimeras Influences Secretase Processing

Amyloid β protein, the major component of the senile plaques in Alzheimer's disease, is generated by secretory and endocytic processing of amyloid precursor protein. Internalized amyloid precursor protein either recycles to the plasma membrane, where α-secretase resides, or moves to acidic compartment(s) for β-secretase exposure. While the trans-Golgi network contains β-secretase activity, recent examination of the subcellular distribution of this proteinase, called BACE, has led to the suggestion that β-secretase activity might also reside at the plasma membrane and in endosomes. To examine the role of endocytic compartments in β-secretase processing of amyloid precursor protein, the wild-type and endosomal sorting mutant P-selectin cytoplasmic domains were used to control movement of amyloid precursor protein through endosomes. Amyloid precursor protein/P-selectin, which is sorted from early to late endosomes, undergoes significantly less α-secretase cleavage, and more β-secretase cleavage, than amyloid precursor protein/P-selectin768A, a mutant that recycles more efficiently to the cell surface. Our results demonstrate that endosomal sorting influences relative exposure of the amyloid precursor protein/P-selectin chimeras to α- and β-secretase activities, and suggest that, because delivery to late endocytic compartments favors β-secretase processing of amyloid precursor protein, there is likely limited β-secretase activity in early endosomes or at the cell surface. We propose that the trans-Golgi network may be involved in both secretory and endocytic generation of amyloid β protein.     

The Two Endocytic Pathways Mediated by the Carbohydrate Recognition Domain and Regulated by the Collagen-like Domain of Galectin-3 in Vascular Endothelial Cells

Galectin-3 plays an important role in endothelial morphogenesis and angiogenesis. We investigated the endocytosis of galectin-3 in human vascular endothelial cells and showed that galectin-3 could associate with and internalized into the cells in a carbohydrate-dependent manner. Our work also revealed that galectin-3 was transported to the early/recycling endosomes and then partitioned into two routes – recycling back to the plasma membrane or targeting to the late endosomes/lysosomes. Various N- and C-terminal truncated forms of galectin-3 were constructed and compared with the full-length protein. These comparisons showed that the carbohydrate-recognition domain of galectin-3 was required for galectin-3 binding and endocytosis. The N-terminal half of the protein, which comprises the N-terminal leader domain and the collagen-like internal repeating domain, could not mediate binding and endocytosis alone. The collagen-like domain, although it was largely irrelevant to galectin-3 trafficking to the early/recycling endosomes, was required for targeting galectin-3 to the late endosomes/lysosomes. In contrast, the leader domain was irrelevant to both binding and intracellular trafficking. The data presented in this study correlate well with different cellular behaviors induced by the full-length and the truncated galectin-3 and provide an alternative way of understanding its angiogenic mechanisms.     

Sorting of the neuroendocrine secretory protein Secretogranin II into the regulated secretory pathway: role of N- and C-terminal alpha-helical domains

Secretogranin II (SgII) belongs to the granin family of prohormones widely distributed in dense-core secretory granules (DCGs) of endocrine, neuroendocrine, and neuronal cells, including sympathoadrenal chromaffin cells. The mechanisms by which secretory proteins, and granins in particular, are sorted into the regulated secretory pathway are unsettled. We designed a strategy based on novel chimeric forms of human SgII fused to fluorescent (green fluorescent protein) or chemiluminescent (embryonic alkaline phosphatase) reporters to identify trafficking determinants mediating DCG targeting of SgII in sympathoadrenal cells. Three-dimensional deconvolution fluorescence microscopy and secretagogue-stimulated release studies demonstrate that SgII chimeras are correctly targeted to DCGs and released by exocytosis in PC12 and primary chromaffin cells. Results from a Golgi-retained mutant form of SgII suggest that sorting of SgII into DCGs depends on a saturable sorting machinery at the trans-Golgi/trans-Golgi network. Truncation analyses reveal the presence of DCG-targeting signals within both the N- and C-terminal regions of SgII, with the putative alpha-helix-containing SgII-(25-41) and SgII-(334-348) acting as sufficient, independent sorting domains. This study defines sequence features of SgII mediating vesicular targeting in sympathoadrenal cells and suggests a mechanism by which discrete domains of the molecule function in sorting, perhaps by virtue of a particular arrangement in tertiary structure and/or interaction with a specific component of the DCG membrane.     

Sorting nexin 17 accelerates internalization yet retards degradation of P-selectin

The transient appearance of P-selectin on the surface of endothelial cells helps recruit leukocytes into sites of inflammation. The tight control of cell surface P-selectin on these cells depends on regulated exocytosis of Weibel-Palade bodies where the protein is stored and on its rapid endocytosis. After endocytosis, P-selectin is either sorted via endosomes and the Golgi apparatus for storage in Weibel-Palade bodies or targeted to lysosomes for degradation. A potential player in this complex endocytic itinerary is SNX17, a member of the sorting nexin family, which has been shown in a yeast two-hybrid assay to bind P-selectin. Here, we show that overexpression of SNX17 in mammalian cells can influence two key steps in the endocytic trafficking of P-selectin. First, it promotes the endocytosis of P-selectin from the plasma membrane. Second, it inhibits the movement of P-selectin into lysosomes, thereby reducing its degradation.