Post-translational N-glycosylation of type I transmembrane KCNE1 peptides: implications for membrane protein biogenesis and disease

N-Glycosylation of membrane proteins is critical for their proper folding, co-assembly and subsequent matriculation through the secretory pathway. Here, we examine the kinetics of N-glycan addition to type I transmembrane KCNE1 K(+) channel β-subunits, where point mutations that prevent N-glycosylation at one consensus site give rise to disorders of the cardiac rhythm and congenital deafness. We show that KCNE1 has two distinct N-glycosylation sites: a typical co-translational site and a consensus site ～20 residues away that unexpectedly acquires N-glycans after protein synthesis (post-translational). Mutations that ablate the co-translational site concomitantly reduce glycosylation at the post-translational site, resulting in unglycosylated KCNE1 subunits that cannot reach the cell surface with their cognate K(+) channel. This long range inhibition is highly specific for post-translational N-glycosylation because mutagenic conversion of the KCNE1 post-translational site into a co-translational site restored both monoglycosylation and anterograde trafficking. These results directly explain how a single point mutation can prevent N-glycan attachment at multiple sites, providing a new biogenic mechanism for human disease.     

Adenosine A2A receptor is involved in cell surface expression of A2B receptor

The A2A and A2B adenosine receptors (A2AR and A2BR) are implicated in many physiological processes. However, the mechanisms of their intracellular maturation and trafficking are poorly understood. In comparative studies of A2AR versus A2BR expression in transfected cells, we noticed that the levels of cell surface expression of A2BR were significantly lower than those of A2AR. A large portion of the A2BR was degraded by the proteasome. Studies of cell surface expression of A2BR chimeric molecules in transfectants suggested that A2BR does not have the dominant forward transport signal for export from the endoplasmic reticulum to the cell surface. A2BR surface expression was increased in A2BR chimeras where the A2BR carboxyl terminus (CT) was replaced or fused with the A2AR CT. Co-transfection of A2AR with A2BR enhanced surface expression of A2BR though the F(X)(6)LL motif in the A2AR CT. The requirements of A2AR expression for better A2BR cell surface expression was not only established in transfectants but also confirmed by observations of much lower levels of A2BR-induced intracellular cAMP accumulation in response to A2BR-activating ligand in splenocytes from A2AR(-/-) mice than in wild type mice. The results of mechanistic studies suggested that poor A2BR expression at the cell surface might be accounted for mainly by the lack of a dominant forward transport signal from the endoplasmic reticulum to the plasma membrane; it is likely that A2BR forms a hetero-oligomer complex for better function.     

Sec23 Homolog Nel1 Is a Novel GTPase-activating Protein for Sar1 but Does Not Function as a Subunit of the Coat Protein Complex II (COPII) Coat

The coat protein complex II (COPII) generates transport carriers from the endoplasmic reticulum (ER) under the control of the small GTPase Sar1. Sec23 is well known as a structural component of the COPII coat and as a GTPase-activating protein (GAP) for Sar1. Here, we showed that Saccharomyces cerevisiae contains a novel Sec23 paralog, Nel1, which appears not to function as a subunit of the COPII coat. Nel1 does not associate with any of the COPII components, but it exhibits strong Sar1 GAP activity. We also demonstrated that the chromosomal deletion of NEL1 leads to a significant growth defect in the temperature-sensitive sar1D32G background, suggesting a possible functional link between these proteins. In contrast to Sec23, which is predominantly localized at ER exit sites on the ER membrane, a major proportion of Nel1 is localized throughout the cytosol. Our findings highlight a possible role of Nel1 as a novel GAP for Sar1.     

Examination of Sec22 Homodimer Formation and Role in SNARE-dependent Membrane Fusion

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein complexes play essential roles in catalyzing intracellular membrane fusion events although the assembly pathway and molecular arrangement of SNARE complexes in membrane fusion reactions are not well understood. Here we monitored interactions of the R-SNARE protein Sec22 through a cysteine scanning approach and detected efficient formation of cross-linked Sec22 homodimers in cellular membranes when cysteine residues were positioned in the SNARE motif or C terminus of the transmembrane domain. When specific Sec22 cysteine derivatives are present on both donor COPII vesicles and acceptor Golgi membranes, the formation of disulfide cross-links provide clear readouts on trans- and cis-SNARE arrangements during this fusion event. The Sec22 transmembrane domain was required for efficient homodimer formation and for membrane fusion suggesting a functional role for Sec22 homodimers. We propose that Sec22 homodimers promote assembly of higher-order SNARE complexes to catalyze membrane fusion. Sec22 is also reported to function in macroautophagy and in formation of endoplasmic reticulum-plasma membrane contact sites therefore homodimer assembly may regulate Sec22 activity across a range of cellular processes.     

Protrudin Regulates Endoplasmic Reticulum Morphology and Function Associated with the Pathogenesis of Hereditary Spastic Paraplegia

Protrudin is a membrane protein that regulates polarized vesicular trafficking in neurons. The protrudin gene (ZFYVE27) is mutated in a subset of individuals with hereditary spastic paraplegia (HSP), and protrudin is therefore also referred to as spastic paraplegia (SPG) 33. We have now generated mice that express a transgene for dual epitope-tagged protrudin under control of a neuron-specific promoter, and we have subjected highly purified protrudin-containing complexes isolated from the brain of these mice to proteomics analysis to identify proteins that associate with protrudin. Protrudin was found to interact with other HSP-related proteins including myelin proteolipid protein 1 (SPG2), atlastin-1 (SPG3A), REEP1 (SPG31), REEP5 (similar to REEP1), Kif5A (SPG10), Kif5B, Kif5C, and reticulon 1, 3, and 4 (similar to reticulon 2, SPG12). Membrane topology analysis indicated that one of three hydrophobic segments of protrudin forms a hydrophobic hairpin domain similar to those of other SPG proteins. Protrudin was found to localize predominantly to the tubular endoplasmic reticulum (ER), and forced expression of protrudin promoted the formation and stabilization of the tubular ER network. The protrudin(G191V) mutant, which has been identified in a subset of HSP patients, manifested an increased intracellular stability, and cells expressing this mutant showed an increased susceptibility to ER stress. Our results thus suggest that protrudin contributes to the regulation of ER morphology and function, and that its deregulation by mutation is a causative defect in HSP.     

Maturation of thyroglobulin protein region I

In vertebrates, the thyroglobulin (Tg) gene product must be exported to the lumen of thyroid follicles for thyroid hormone synthesis. In toto, Tg is composed of multiple type-1 repeats connected by linker and hinge (altogether considered as region I, nearly 1,200 residues); regions II-III (~720 residues); and cholinesterase-like (ChEL) domain (~570 residues). Regions II-III and ChEL rapidly acquire competence for secretion, yet regions I-II-III require 20 min to become a partially mature disulfide isomer; stabilization of a fully oxidized form requires ChEL. Transition from partially mature to mature Tg occurs as a discrete jump in mobility by nonreducing SDS-PAGE, suggesting formation of at most a few final pairings of Cys residues that may be separated by significant intervening primary sequence. Using two independent approaches, we have investigated which portion of Tg is engaged in this late stage of its maturation. First, we demonstrate that this event is linked to oxidation involving region I. Introduction of the Tg-C1245R mutation in the hinge (identical to that causing human goitrous hypothyroidism) inhibits this maturation, although the Cys-1245 partner remains unidentified. Second, we find that Tg truncated after its fourth type-1 repeat is a fully independent secretory protein. Together, the data indicate that final acquisition of secretory competence includes conformational maturation in the interval between linker and hinge segments of region I.     

Silencing of the Tandem Pore Domain Halothane-inhibited K+ Channel 2 (THIK2) Relies on Combined Intracellular Retention and Low Intrinsic Activity at the Plasma Membrane

The tandem pore domain halothane-inhibited K⁺ channel 1 (THIK1) produces background K⁺ currents. Despite 62% amino acid identity with THIK1, THIK2 is not active upon heterologous expression. Here, we show that this apparent lack of activity is due to a unique combination of retention in the endoplasmic reticulum and low intrinsic channel activity at the plasma membrane. A THIK2 mutant containing a proline residue (THIK2-A155P) in its second inner helix (M2) produces K⁺-selective currents with properties similar to THIK1, including inhibition by halothane and insensitivity to extracellular pH variations. Another mutation in the M2 helix (I158D) further increases channel activity and affects current kinetics. We also show that the cytoplasmic amino-terminal region of THIK2 (Nt-THIK2) contains an arginine-rich motif (RRSRRR) that acts as a retention/retrieval signal. Mutation of this motif in THIK2 induces a relocation of the channel to the plasma membrane, resulting in measurable currents, even in the absence of mutations in the M2 helix. Cell surface delivery of a Nt-THIK2-CD161 chimera is increased by mutating the arginines of the retention motif but also by converting the serine embedded in this motif to aspartate, suggesting a phosphorylation-dependent regulation of THIK2 trafficking.     

Endoplasmic Reticulum (ER) Localization Is Critical for DsbA-L Protein to Suppress ER Stress and Adiponectin Down-regulation in Adipocytes

Adiponectin is an adipokine with insulin-sensitizing and anti-inflammatory functions. We previously reported that adiponectin multimerization and stability are promoted by the disulfide bond A oxidoreductase-like protein (DsbA-L) in cells and in vivo. However, the precise mechanism by which DsbA-L regulates adiponectin biosynthesis remains elusive. Here we show that DsbA-L is co-localized with the endoplasmic reticulum (ER) marker protein disulfide isomerase and the mitochondrial marker MitoTracker. In addition, DsbA-L interacts with the ER chaperone protein Ero1-Lα in 3T3-L1 adipocytes. In silico analysis and truncation mapping studies revealed that DsbA-L contains an ER targeting signal at its N terminus. Deletion of the first 6 residues at the N terminus greatly impaired DsbA-L localization in the ER. Overexpression of the wild type but not the ER localization-defective mutant of DsbA-L protects against thapsigargin-induced ER stress and adiponectin down-regulation in 3T3-L1 adipocytes. In addition, overexpression of the wild type but not the ER localization-defective mutant of DsbA-L promotes adiponectin multimerization. Together, our results reveal that DsbA-L is localized in both the mitochondria and the ER in adipocytes and that its ER localization plays a critical role in suppressing ER stress and promoting adiponectin biosynthesis and secretion.     

Mammalian COPII Coat Component SEC24C Is Required for Embryonic Development in Mice

COPII-coated vesicles mediate the transport of newly synthesized proteins from the endoplasmic reticulum to the Golgi. SEC24 is the COPII component primarily responsible for recruitment of protein cargoes into nascent vesicles. There are four Sec24 paralogs in mammals, with mice deficient in SEC24A, -B, and -D exhibiting a wide range of phenotypes. We now report the characterization of mice with deficiency in the fourth Sec24 paralog, SEC24C. Although mice haploinsufficient for Sec24c exhibit no apparent abnormalities, homozygous deficiency results in embryonic lethality at approximately embryonic day 7. Tissue-specific deletion of Sec24c in hepatocytes, pancreatic cells, smooth muscle cells, and intestinal epithelial cells results in phenotypically normal mice. Thus, SEC24C is required in early mammalian development but is dispensable in a number of tissues, likely as a result of compensation by other Sec24 paralogs. The embryonic lethality resulting from loss of SEC24C occurs considerably later than the lethality previously observed in SEC24D deficiency; it is clearly distinct from the restricted neural tube phenotype of Sec24b null embryos and the mild hypocholesterolemic phenotype of adult Sec24a null mice. Taken together, these results demonstrate that the four Sec24 paralogs have developed unique functions over the course of vertebrate evolution.     

Transmembrane Segments Prevent Surface Expression of Sodium Channel Nav1.8 and Promote Calnexin-dependent Channel Degradation

The voltage-gated sodium channel (Na_v) 1.8 contributes substantially to the rising phase of action potential in small dorsal root ganglion neurons. Na_v1.8 is majorly localized intracellularly and its expression on the plasma membrane is regulated by exit from the endoplasmic reticulum (ER). Previous work has identified an ER-retention/retrieval motif in the first intracellular loop of Na_v1.8, which prevents its surface expression. Here we report that the transmembrane segments of Na_v1.8 also cause this channel retained in the ER. Using transferrin receptor and CD8α as model molecules, immunocytochemistry showed that the first, second, and third transmembrane segments in each domain of Na_v1.8 reduced their surface expression. Alanine-scanning analysis revealed acidic amino acids as critical factors in the odd transmembrane segments. Furthermore, co-immunoprecipitation experiments showed that calnexin interacted with acidic amino acid-containing sequences through its transmembrane segment. Overexpression of calnexin resulted in increased degradation of those proteins through the ER-associated degradation pathway, whereas down-regulation of calnexin reversed the phenotype. Thus our results reveal a critical role and mechanism of transmembrane segments in surface expression and degradation of Na_v1.8.     

Apoptosis-linked Gene-2 (ALG-2)/Sec31 Interactions Regulate Endoplasmic Reticulum (ER)-to-Golgi Transport: A POTENTIAL EFFECTOR PATHWAY FOR LUMINAL CALCIUM*

Luminal calcium released from secretory organelles has been suggested to play a regulatory role in vesicle transport at several steps in the secretory pathway; however, its functional roles and effector pathways have not been elucidated. Here we demonstrate for the first time that specific luminal calcium depletion leads to a significant decrease in endoplasmic reticulum (ER)-to-Golgi transport rates in intact cells. Ultrastructural analysis revealed that luminal calcium depletion is accompanied by increased accumulation of intermediate compartment proteins in COPII buds and clusters of unfused COPII vesicles at ER exit sites. Furthermore, we present several lines of evidence suggesting that luminal calcium affected transport at least in part through calcium-dependent interactions between apoptosis-linked gene-2 (ALG-2) and the Sec31A proline-rich region: 1) targeted disruption of ALG-2/Sec31A interactions caused severe defects in ER-to-Golgi transport in intact cells; 2) effects of luminal calcium and ALG-2/Sec31A interactions on transport mutually required each other; and 3) Sec31A function in transport required luminal calcium. Morphological phenotypes of disrupted ALG-2/Sec31A interactions were characterized. We found that ALG-2/Sec31A interactions were not required for the localization of Sec31A to ER exit sites per se but appeared to acutely regulate the stability and trafficking of the cargo receptor p24 and the distribution of the vesicle tether protein p115. These results represent the first outline of a mechanism that connects luminal calcium to specific protein interactions regulating vesicle trafficking machinery.     

The NPC motif of aquaporin-11, unlike the NPA motif of known aquaporins, is essential for full expression of molecular function

The recently identified molecule aquaporin-11 (AQP11) has a unique amino acid sequence pattern that includes an Asn-Pro-Cys (NPC) motif, corresponding to the N-terminal Asn-Pro-Ala (NPA) signature motif of conventional AQPs. In this study, we examined the effect of the mutation of the NPC motif on the subcellular localization, oligomerization, and water permeability of AQP11 in transfected mammalian cells. Furthermore, the effect was also assessed using zebrafish. Site-directed mutation at the NPC motif did not affect the subcellular localization of AQP11 but reduced its oligomerization. A cell swelling assay revealed that cells expressing AQP11 with a mutated NPC motif had significantly lower osmotic water permeability than cells expressing wild-type AQP11. Zebrafish deficient in endogenous AQP11 showed a deformity in the tail region at an early stage of development. This phenotype was dramatically rescued by injection of human wild-type AQP11 mRNA, whereas the effect of mRNA for AQP11 with a mutated NPC motif was less marked. Although the NPA motif is known to be important for formation of water-permeable pores by conventional AQPs, our observations suggest that the corresponding NPC motif of AQP11 is essential for full expression of molecular function.     

Repeat motif-containing regions within thyroglobulin

Thyroglobulin (precursor for thyroid hormone synthesis) is a large secreted glycoprotein comprising contiguous region I (multiple type-1 repeating units engaging the first ～1,191 residues, followed by a ～245-residue hinge region), regions II-III (multiple type-2 and 3 repeating units, comprising ～720 residues), and the C-terminal cholinesterase-like (ChEL) domain (～570 residues). A signal peptide attached to ChEL makes an independent secretory protein that binds to I-II-III, stabilizing it and rescuing the secretion of I-II-III that would otherwise be trapped in the endoplasmic reticulum (ER). In this study, we found that a signal peptide attached to regions II-III also makes for an efficient secretory protein that neither demonstrably interacts nor has its secretion enhanced by the presence of secretory ChEL. By contrast, region I, either with or without the hinge region, cannot be secreted on its own and remains in the ER where it is bound to ER chaperones BiP and GRP94. Whereas ChEL can rescue secretion of I-II-III, it can rescue I-II only very weakly, and region I not at all. Yet, ChEL begins to rescue region I in cells that also co-express secretory II-III. The data suggest that conformational maturation of region I is a limiting step in the thyroglobulin maturation process, and this step is facilitated by the presence of both regions II-III and ChEL. Mutations causing hypothyroidism might induce solely local/regional misfolding or may interfere more globally by impeding interactions between regions that are required for thyroglobulin secretion.     

TMTC1 and TMTC2 Are Novel Endoplasmic Reticulum Tetratricopeptide Repeat-containing Adapter Proteins Involved in Calcium Homeostasis

The endoplasmic reticulum (ER) is organized in part by adapter proteins that nucleate the formation of large protein complexes. Tetratricopeptide repeats (TPR) are well studied protein structural motifs that support intermolecular protein-protein interactions. TMTC1 and TMTC2 were identified by an in silico search as TPR-containing proteins possessing N-terminal ER targeting signal sequences and multiple hydrophobic segments, suggestive of polytopic membrane proteins that are targeted to the secretory pathway. A variety of cell biological and biochemical assays was employed to demonstrate that TMTC1 and TMTC2 are both ER resident integral membrane proteins with multiple clusters of TPR domains oriented within the ER lumen. Proteomic analysis followed by co-immunoprecipitation verification found that both proteins associated with the ER calcium uptake pump SERCA2B, and TMTC2 also bound to the carbohydrate-binding chaperone calnexin. Live cell calcium measurements revealed that overexpression of either TMTC1 or TMTC2 caused a reduction of calcium released from the ER following stimulation, whereas the knockdown of TMTC1 or TMTC2 increased the stimulated calcium released. Together, these results implicate TMTC1 and TMTC2 as ER proteins involved in ER calcium homeostasis.     

Mutations in the carboxyl-terminal SEC24 binding motif of the serotonin transporter impair folding of the transporter

The serotonin transporter (SERT) is a member of the SLC6 family of solute carriers. SERT plays a crucial role in synaptic neurotransmission by retrieving released serotonin. The intracellular carboxyl terminus of various neurotransmitter transporters has been shown to be important for the correct delivery of SLC6 family members to the cell surface. Here we studied the importance of the C terminus in trafficking and folding of human SERT. Serial truncations followed by mutagenesis identified sequence spots (PG(601,602), RII(607-609)) within the C terminus relevant for export of SERT from the endoplasmic reticulum (ER). RI(607,608) is homologous to the RL-motif that in other SLC6 family members provides a docking site for the COPII component Sec24D. The primary defect resulting from mutation at PG(601,602) and RI(607,608) was impaired folding, because mutated transporters failed to bind the inhibitor [(3)H]imipramine. In contrast, when retained in the ER (e.g. by dominant negative Sar1) the wild type transporter bound [(3)H]imipramine with an affinity comparable to that of the surface-expressed transporter. SERT-RI(607,608)AA and SERT-RII(607-609)AAA were partially rescued by treatment of cells with the nonspecific chemical chaperone DMSO or the specific pharmacochaperone ibogaine (which binds to the inward facing conformation of SERT) but not by other classes of ligands (inhibitors, substrates, amphetamines). These observations (i) demonstrate an hitherto unappreciated role of the C terminus in the folding of SERT, (ii) indicates that the folding trajectory proceeds via an inward facing intermediate, and (iii) suggest a model where the RI-motif plays a crucial role in preventing premature Sec24-recruitment and export of incorrectly folded transporters.     

Isoform-selective Oligomer Formation of Saccharomyces cerevisiae p24 Family Proteins

p24 family proteins are evolutionarily conserved transmembrane proteins involved in the early secretory pathway. Saccharomyces cerevisiae has 8 known p24 proteins that are classified into four subfamilies (p24α, -β, -γ, and -δ). Emp24 and Erv25 are the sole members of p24β and -δ, respectively, and deletion of either destabilizes the remaining p24 proteins, resulting in p24 null phenotype (p24Δ). We studied genetic and physical interactions of p24α (Erp1, -5, and -6) and γ (Erp2, -3, and -4). Deletion of the major p24α (Erp1) partially inhibited p24 activity as reported previously. A second mutation in either Erp5 or Erp6 aggravated the erp1Δ phenotype, and the triple mutation gave a full p24Δ phenotype. Similar genetic interactions were observed among the major p24γ (Erp2) and the other two γ members. All the p24α/γ isoforms interacted with both p24β and -δ. Interaction between p24β and -δ was isoform-selective, and five major α/γ pairs were detected. These results suggest that the yeast p24 proteins form functionally redundant αβγδ complexes. We also identified Rrt6 as a novel p24δ isoform. Rrt6 shows only limited sequence identity (∼15%) to known p24 proteins but was found to have structural properties characteristic of p24. Rrt6 was induced when cells were grown on glycerol and form an additional αβγδ complex with Erp3, Erp5, and Emp24. This complex was mainly localized to the Golgi, whereas the p24 complex containing Erv25, instead of Rrt6 but otherwise with the same isoform composition, was found mostly in the ER.     

Carrier subunit of plasma membrane transporter is required for oxidative folding of its helper subunit

We study the amino acid transport system b(0,+) as a model for folding, assembly, and early traffic of membrane protein complexes. System b(0,+) is made of two disulfide-linked membrane subunits: the carrier, b(0,+) amino acid transporter (b(0,+)AT), a polytopic protein, and the helper, related to b(0,+) amino acid transporter (rBAT), a type II glycoprotein. rBAT ectodomain mutants display folding/trafficking defects that lead to type I cystinuria. Here we show that, in the presence of b(0,+)AT, three disulfides were formed in the rBAT ectodomain. Disulfides Cys-242-Cys-273 and Cys-571-Cys-666 were essential for biogenesis. Cys-673-Cys-685 was dispensable, but the single mutants C673S, and C685S showed compromised stability and trafficking. Cys-242-Cys-273 likely was the first disulfide to form, and unpaired Cys-242 or Cys-273 disrupted oxidative folding. Strikingly, unassembled rBAT was found as an ensemble of different redox species, mainly monomeric. The ensemble did not change upon inhibition of rBAT degradation. Overall, these results indicated a b(0,+)AT-dependent oxidative folding of the rBAT ectodomain, with the initial and probably cotranslational formation of Cys-242-Cys-273, followed by the oxidation of Cys-571-Cys-666 and Cys-673-Cys-685, that was completed posttranslationally.     

[Ca2+]i Elevation and Oxidative Stress Induce KCNQ1 Protein Translocation from the Cytosol to the Cell Surface and Increase Slow Delayed Rectifier (IKs) in Cardiac Myocytes

Our goals are to simultaneously determine the three-dimensional distribution patterns of KCNQ1 and KCNE1 in cardiac myocytes and to study the mechanism and functional implications for variations in KCNQ1/KCNE1 colocalization in myocytes. We monitored the distribution patterns of KCNQ1, KCNE1, and markers for subcellular compartments/organelles using immunofluorescence/confocal microscopy and confirmed the findings in ventricular myocytes by directly observing fluorescently tagged KCNQ1-GFP and KCNE1-dsRed expressed in these cells. We also monitored the effects of stress on KCNQ1-GFP and endoplasmic reticulum (ER) remodeling during live cell imaging. The data showed that 1) KCNE1 maintained a stable cell surface localization, whereas KCNQ1 exhibited variations in the cytosolic compartment (striations versus vesicles) and the degree of presence on the cell surface; 2) the degree of cell surface KCNQ1/KCNE1 colocalization was positively correlated with slow delayed rectifier (I_Ks) current density; 3) KCNQ1 and calnexin (an ER marker) shared a cytosolic compartment; and 4) in response to stress ([Ca²⁺]_i elevation, oxidative overload, or AT1R stimulation), KCNQ1 exited the cytosolic compartment and trafficked to the cell periphery in vesicles. This was accompanied by partial ER fragmentation. We conclude that the cellular milieu regulates KCNQ1 distribution in cardiac myocytes and that stressful conditions can increase I_Ks by inducing KCNQ1 movement to the cell surface. This represents a hitherto unrecognized mechanism by which I_Ks fulfills its function as a repolarization reserve in ventricular myocytes.     

A New Role for Annexin A11 in the Early Secretory Pathway via Stabilizing Sec31A Protein at the Endoplasmic Reticulum Exit Sites (ERES)

Exit of cargo molecules from the endoplasmic reticulum (ER) for transport to the Golgi is the initial step in intracellular vesicular trafficking. The coat protein complex II (COPII) machinery is recruited to specialized regions of the ER, called ER exit sites (ERES), where it plays a central role in the early secretory pathway. It has been known for more than two decades that calcium is an essential factor in vesicle trafficking from the ER to Golgi apparatus. However, the role of calcium in the early secretory pathway is complicated and poorly understood. We and others previously identified Sec31A, an outer cage component of COPII, as an interacting protein for the penta-EF-hand calcium-binding protein ALG-2. In this study, we show that another calcium-binding protein, annexin A11 (AnxA11), physically associates with Sec31A by the adaptor function of ALG-2. Depletion of AnxA11 or ALG-2 decreases the population of Sec31A that is stably associated with the ERES and causes scattering of juxtanuclear ERES to the cell periphery. The synchronous ER-to-Golgi transport of transmembrane cargoes is accelerated in AnxA11- or ALG-2-knockdown cells. These findings suggest that AnxA11 maintains architectural and functional features of the ERES by coordinating with ALG-2 to stabilize Sec31A at the ERES.     

The α-Helical Region in p24γ2 Subunit of p24 Protein Cargo Receptor Is Pivotal for the Recognition and Transport of Glycosylphosphatidylinositol-anchored Proteins

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are group of proteins that depend on p24 cargo receptors for their transport from the endoplasmic reticulum to the Golgi apparatus. The GPI anchor is expected to act as a sorting and transport signal, but so far little is known about the recognition mechanism. In the present study we investigate the GPI-AP transport in cell knockdown of p24γ, the most diverse p24 subfamily. Knockdown of p24γ₂ but not of other p24γ family members impaired the transport of a reporter GPI-AP. Restoration of the knockdown-induced phenotype using chimeric constructs between p24γ₂ and the related p24γ₁ further implied a role of the α-helical region of p24γ₂ but not its GOLD domain in the specific binding of GPI-APs. We conclude that motifs in the membrane-adjacent α-helical region of p24γ₂ are involved in recognition of GPI-APs and are consequently responsible for the incorporation of these proteins into coat protein complex II-coated transport vesicles.