Search

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel in the plasma membrane of several epithelial cells. Maturation of CFTR is inefficient in most cells, with only a fraction of nascent chains being properly folded and transported to the cell surface. The most common mutation in CFTR, CFTR-deltaF508, leads to the genetic disease cystic fibrosis. CFTR-deltaF508 has a temperature-sensitive folding defect and is almost quantitatively degraded in the endoplasmic reticulum (ER). Here we tested whether a strong ER export signal appended to CFTR improves its transport and surface expression. We show that a single valine ER export signal at the C terminus of the cytoplasmic tail of CFTR improves maturation of wild-type CFTR by 2-fold. This conservative mutation interfered with neither plasma membrane localization nor stability of mature CFTR. In contrast, the valine signal was unable to rescue CFTR-deltaF508 from ER-associated degradation. Our finding of improved maturation of CFTR mediated by a valine signal may be of potential use in gene therapy of cystic fibrosis. Moreover, failure of the valine signal to rescue CFTR-deltaF508 from ER degradation indicates that the inability of CFTR-deltaF508 to leave the ER is unlikely to be due to a malfunctioning ER export signal.

The recycling mannose lectin ERGIC-53 operates as a transport receptor by mediating efficient endoplasmic reticulum (ER) export of some secretory glycoproteins. Binding of cargo to ERGIC-53 in the ER requires Ca2+. Cargo release occurs in the ERGIC, but the molecular mechanism is unknown. Here we report efficient binding of purified ERGIC-53 to immobilized mannose at pH 7.4, the pH of the ER, but not at slightly lower pH. pH sensitivity of the lectin was more prominent when Ca2+ concentrations were low. A conserved histidine in the center of the carbohydrate recognition domain was required for lectin activity suggesting it may serve as a molecular pH/Ca2+ sensor. Acidification of cells inhibited the association of ERGIC-53 with the known cargo cathepsin Z-related protein and dissociation of this glycoprotein in the ERGIC was impaired by organelle neutralization that did not impair the transport of a control protein. The results elucidate the molecular mechanism underlying reversible lectin/cargo interaction and establish the ERGIC as the earliest low pH site of the secretory pathway.

Selective export of proteins from the endoplasmic reticulum (ER) requires transport signals that have not been fully characterized. Here, we provide the first complete map of ER export determinants of a type I membrane protein, ERGIC-53, that cycles in the early secretory pathway. ER export requires a phenylalanine motif at the C-terminus, known to mediate coat protein II (COPII) interaction, that is assisted by a glutamine in the cytoplasmic domain. Disulfide bond-stabilized oligomerization is also required. Efficient hexamerization depends on the presence of a polar and two aromatic residues in the transmembrane domain (TMD). Oligomerization becomes independent on disulfide bonds when TMD hydrophobicity is increased. ER export is also influenced by TMD length, 21 amino acids being most efficient. When transferred to a signal-less construct, the established targeting motifs reconstitute full transport activity. The results suggest an ER-export mechanism in which transmembrane and luminal determinants mediate oligomerization required for efficient recruitment of ERGIC-53 into budding vesicles via the C-terminal COPII-binding phenylalanine motif.

The accumulation of unfolded proteins in the ER triggers a signaling response known as unfolded protein response (UPR). In yeast the UPR affects several hundred genes that encode ER chaperones and proteins operating at later stages of secretion. In mammalian cells the UPR appears to be more limited to chaperones of the ER and genes assumed to be important after cell recovery from ER stress that are not important for secretion. Here, we report that the mRNA of lectin ERGIC-53, a cargo receptor for the transport of glycoproteins from ER to ERGIC, and of its related protein VIP36 is induced by the known inducers of ER stress, tunicamycin and thapsigargin. In parallel, the rate of synthesis of the ERGIC-53 protein was induced by these agents. The response was due to the UPR since it was also triggered by castanospermine, a specific inducer of UPR, and inhibited by genistein. Thapsigargin-induced upregulation of ERGIC-53 could be fully accounted for by the ATF6 pathway of UPR. The results suggest that in mammalian cells the UPR also affects traffic from and beyond the ER.

Forward transport of proteins from the ER to the plasma membrane requires escape from the ER's retention machinery. Recent studies suggest that 14-3-3 proteins may mediate ER export of potassium channels destined for the plasma membrane by interfering with dibasic-motif-mediated retention.

Consensus profiles were established to screen data bases for novel animal L-type lectins. The profiles were generated from linear sequence motifs of the human L-type lectin-like membrane proteins ERGIC-53, ERGL, and VIP36 and by optimal alignment of the entire carbohydrate recognition domain of these proteins. The search revealed numerous orthologous and homologous L-type lectin-like proteins in animals, protozoans, and yeast, as well as the sequence of a novel family member related to VIP36, named VIPL for VIP36-like. Sequence analysis suggests that VIPL is a ubiquitously expressed protein and appeared earlier in evolution than VIP36. The cDNA of VIPL was cloned and expressed in cell culture. VIPL is a high-mannose type I membrane glycoprotein with similar domain organization as VIP36. Unlike VIP36 and ERGIC-53 that are predominantly associated with postendoplasmic reticulum (ER) membranes and cycle in the early secretory pathway, VIPL is a non-cycling resident protein of the ER. Mutagenesis experiments indicate that ER retention of VIPL involves a RKR di-arginine signal. Overexpression of VIPL redistributed ERGIC-53 to the ER without affecting the cycling of the KDEL-receptor and the overall morphology of the early secretory pathway. The results suggest that VIPL may function as a regulator of ERGIC-53.

Export of membrane proteins from the ER is believed to be selective and require transport signals, but the identity of such signals has remained elusive. The recycling type I membrane protein ERGIC-53 carries a C-terminal diphenylalanine motif that is required for efficient ER export. Here we show that this motif can be functionally substituted by a single phenylalanine or tyrosine at position -2, two leucines or isoleucines at position -1 and -2 or a single valine at position -1. These motifs are common among mammalian type I membrane proteins. A single C-terminal valine, but none of the other motifs, accelerates transport of inefficiently exported reporter constructs and hence operates as an export signal. The valine signal is position, but not context, dependent. All transport motifs mediate COPII binding in vitro with distinct preferences for the COPII subunits Sec23p, Sec24Bp, Sec24Cp and p125. These results suggest that cytoplasmic C-terminal amino-acid motifs, either alone or in conjunction with other transport determinants, accelerate ER export of numerous type I and probably polytopic membrane proteins by mediating interaction with COPII coat components.

Lectins of the early secretory pathway are involved in selective transport of newly synthesized glycoproteins from the endoplasmic reticulum (ER) to the ER-Golgi intermediate compartment (ERGIC). The most prominent cycling lectin is the mannose-binding type I membrane protein ERGIC-53 (ERGIC protein of 53 kDa), a marker for the ERGIC, which functions as a cargo receptor to facilitate export of an increasing number of glycoproteins with different characteristics from the ER. Two ERGIC-53-related proteins, VIP36 (vesicular integral membrane protein 36) and a novel ERGIC-53-like protein, ERGL, are also found in the early secretory pathway. ERGL may act as a regulator of ERGIC-53. Studies of ERGIC-53 continue to provide new insights into the organization and dynamics of the early secretory pathway. Analysis of the cycling of ERGIC-53 uncovered a complex interplay of trafficking signals and revealed novel cytoplasmic ER-export motifs that interact with COP-II coat proteins. These motifs are common to type I and polytopic membrane proteins including presenilin 1 and presenilin 2. The results support the notion that protein export from the ER is selective.

Epithelial cell-derived neutrophil-activating protein-78 (ENA-78) is a potent stimulator of neutrophils, inducing a variety of biological responses such as chemotaxis, enzyme release, up-regulation of surface receptors, and intracellular calcium mobilization. Proteolysis of ENA-78 with cathepsin G and chymotrypsin yielded a time-dependent increase in elastase-releasing activity, predicting the formation of truncation products with higher potency than native ENA-78. To investigate the biological implications of progressive truncation of ENA-78, the N-terminal variants ENA(5-78), ENA(9-78), and ENA(10-78) were cloned and expressed in E. coli. When tested in the neutrophil elastase release assay, the variants ENA(5-78) and ENA(9-78) had a 2-3-fold higher potency than full-length ENA-78, while ENA(10-78) was 3-fold less potent. In the chemotaxis assay, the variant ENA(5-78) exhibited an 8-fold and ENA(9-78) a 2-fold higher potency than native ENA-78. ENA(10-78), conversely, was 10-fold less potent, but reached a comparable efficacy to ENA-78 at 10(-)7 M concentration. In summary, the rank order in potency with respect to elastase release was ENA(9-78) > ENA(5-78) > ENA-78 > ENA(10-78), while for chemotaxis it was ENA(5-78) > ENA(9-78) > ENA-78 > ENA(10-78). Variant ENA(5-78) had a higher overall potency and efficiency for chemotaxis than interleukin-8 (IL-8), while ENA(9-78) exhibited a higher efficiency at concentrations of 1-100 nM. The fact that neutrophil cathepsin G produces the stable ENA(9-78) variant in vitro strongly suggests a role for this N-terminal proteolysis during inflammatory processes in vivo.