Endoplasmic Reticulum In HeLa Cells Research Articles

A simple benzothiazole-based turn-on fluorescent probe for detecting H2S in HeLa cells

H2S is an endogenously produced gaseous signaling molecule that plays a vital role in many biological processes. Small molecule fluorescent probes as a powerful tool can be used to in vivo and in vitro visualize the H2S with many advantages. At present, many fluorescence probes were reported based on the nucleophilicity of H2S, which were susceptible to interference from other nucleophilic species and possessed poor selectivity. In this work, we designed a simple benzothiazole-based turn-on fluorescence probe HBT-PBA that was modified with pyridyl disulfide as a recognition group and had good selectivity towards H2S over biothiols. Additionally, HBT-PBA was utilized for fluorescence images of the H2S in the lysosomes and endoplasmic reticulum of HeLa cells.

A novel AIRE-based fluorescent ratiometric probe with endoplasmic reticulum-targeting ability for detection of hypochlorite and bioimaging

Hypochlorite (ClO−) plays an important role in the human immune defense system, but high concentrations of ClO− in the endoplasmic reticulum (ER) damage cellular proteins, causing ER stress, cell death, and various diseases. Herein, we developed a simple hydrazone probe (1) featuring aggregation-induced ratiometric emission, which would quickly (within 20 s) and sensitively (detection limit of 15.4 μM) respond to ClO− in an almost pure aqueous solution via a fluorescent ratiometric output. Furthermore, the probe was employed to track the level of ClO− in the ER of HeLa cells and zebrafish.

Single-cell temperature mapping with fluorescent thermometer nanosheets.

Recent studies using intracellular thermometers have shown that the temperature inside cultured single cells varies heterogeneously on the order of 1°C. However, the reliability of intracellular thermometry has been challenged both experimentally and theoretically because it is, in principle, exceedingly difficult to exclude the effects of nonthermal factors on the thermometers. To accurately measure cellular temperatures from outside of cells, we developed novel thermometry with fluorescent thermometer nanosheets, allowing for noninvasive global temperature mapping of cultured single cells. Various types of cells, i.e., HeLa/HEK293 cells, brown adipocytes, cardiomyocytes, and neurons, were cultured on nanosheets containing the temperature-sensitive fluorescent dye europium (III) thenoyltrifluoroacetonate trihydrate. First, we found that the difference in temperature on the nanosheet between nonexcitable HeLa/HEK293 cells and the culture medium was less than 0.2°C. The expression of mutated type 1 ryanodine receptors (R164C or Y523S) in HEK293 cells that cause Ca2+ leak from the endoplasmic reticulum did not change the cellular temperature greater than 0.1°C. Yet intracellular thermometry detected an increase in temperature of greater than ∼2°C at the endoplasmic reticulum in HeLa cells upon ionomycin-induced intracellular Ca2+ burst; global cellular temperature remained nearly constant within ±0.2°C. When rat neonatal cardiomyocytes or brown adipocytes were stimulated by a mitochondrial uncoupling reagent, the temperature was nearly unchanged within ±0.1°C. In cardiomyocytes, the temperature was stable within ±0.01°C during contractions when electrically stimulated at 2 Hz. Similarly, when rat hippocampal neurons were electrically stimulated at 0.25 Hz, the temperature was stable within ±0.03°C. The present findings with nonexcitable and excitable cells demonstrate that heat produced upon activation in single cells does not uniformly increase cellular temperature on a global basis, but merely forms a local temperature gradient on the order of ∼1°C just proximal to a heat source, such as the endoplasmic/sarcoplasmic reticulum ATPase.

Lipid Nanoparticle-Mediated Induction of Endoplasmic Reticulum Stress in Cancer Cells

The endoplasmic reticulum (ER) primarily guides protein synthesis, folding, transport, and lipid biosynthesis inside the cells. As a result, dysregulation in those cellular functions leading to ER stress has recently emerged as one of the hallmarks of cancer. Yet, precise navigation in the ER in cancer cells has continued to be a formidable task. Herein, we engineered a lipid nanoparticle (17AAG-ER-NP) containing (a) ER targeting moiety (Tosyl), (b) fluorescent tag with DNA damaging capability (1,8-naphthalimide), and (c) ER stress inducer (17AAG, Hsp90 inhibitor). These lipidic nanoparticles were confined in the ER of HeLa cells over 6 h through caveolin-controlled endocytosis confirmed by confocal microscopy. Western blot analysis, fluorescent microscopy, and flow cytometry studies confirmed that 17AAG-ER-NPs can concurrently activate ER stress and nuclear DNA impairment for arresting the cell cycle in the G2-M phase to elicit late apoptosis, followed by cell death, in a greatly augmented manner compared to free drugs. Interestingly, this nanoparticle-mediated ER stress activated autophagy, which was suppressed through a cocktail treatment with 17AAG-ER-NPs and chloroquine (autophagy inhibitor), prompted remarkable HeLa cell killing at submicromolar concentration. This nanoplatform can support new tools to impair multiple targets in the ER for future cancer therapy.

Caffeine chelates calcium in the lumen of the endoplasmic reticulum.

Cytosolic Ca2+ signals are often amplified by massive calcium release from the endoplasmic reticulum (ER). This calcium-induced calcium release (CICR) occurs by activation of an ER Ca2+ channel, the ryanodine receptor (RyR), which is facilitated by both cytosolic- and ER Ca2+ levels. Caffeine sensitizes RyR to Ca2+ and promotes ER Ca2+ release at basal cytosolic Ca2+ levels. This outcome is frequently used as a readout for the presence of CICR. By monitoring ER luminal Ca2+ with the low-affinity genetic Ca2+ probe erGAP3, we find here that application of 50 mM caffeine rapidly reduces the Ca2+ content of the ER in HeLa cells by ∼50%. Interestingly, this apparent ER Ca2+ release does not go along with the expected cytosolic Ca2+ increase. These results can be explained by Ca2+ chelation by caffeine inside the ER. Ca2+-overloaded mitochondria also display a drop of the matrix Ca2+ concentration upon caffeine addition. In contrast, in the cytosol, with a low free Ca2+ concentration (10-7 M), no chelation is observed. Expression of RyR3 sensitizes the responses to caffeine with effects both in the ER (increase in Ca2+ release) and in the cytosol (increase in Ca2+ peak) at low caffeine concentrations (0.3-1 mM) that have no effects in control cells. Our results illustrate the fact that simultaneous monitoring of both cytosolic- and ER Ca2+ are necessary to understand the action of caffeine and raise concerns against the use of high concentrations of caffeine as a readout of the presence of CICR.

Monitoring Changes in the Oligomeric State of a Candidate Endoplasmic Reticulum (ER) Ceramide Sensor by Single-molecule Photobleaching

Single-molecule photobleaching has emerged as a powerful non-invasive approach to extract the stoichiometry of multimeric membrane proteins in their native cellular environment. However, this method has mainly been used to determine the subunit composition of ion channels and receptors at the plasma membrane. Here, we applied single-molecule photobleaching to analyze the oligomeric state of an endoplasmic reticulum (ER) resident candidate ceramide sensor protein, SMSr/SAMD8. Co-immunoprecipitation and chemical cross-linking studies previously revealed that the N-terminal sterile alpha motif (or SAM) domain of SMSr drives self-assembly of the protein into oligomers and that SMSr oligomerization is promoted by curcumin, a drug known to perturb ER ceramide and calcium homeostasis. Application of cell spreading surface-active coating materials in combination with total internal reflection fluorescence (TIRF) microscopy allowed us to image GFP-tagged SMSr proteins as single fluorescent spots in the ER of HeLa cells in which expression of endogenous SMSr was abolished. In line with our biochemical analysis, we find that the number of bleaching steps in SMSr-GFP-positive spots displays a substantial drop after removal of the SAM domain. In contrast, treatment of cells with curcumin increased the number of bleaching steps. Our results document the first successful application of single-molecule photobleaching to resolve drug-induced and domain-dependent changes in the oligomeric state of an ER-resident membrane protein, hence establishing a complementary method to unravel the mechanism by which SMSr controls ceramide levels in the ER.

Development of a stable ERroGFP variant suitable for monitoring redox dynamics in the ER.

The endoplasmic reticulum (ER) is an essential organelle for cellular metabolic homeostasis including folding and maturation of secretory and membrane proteins. Disruption of ER proteostasis has been implicated in the pathogenesis of various diseases such as diabetes and neurodegenerative diseases. The ER redox state, which is an oxidative environment suitable for disulfide-bond formation, is essential for ER protein quality control. Hence, detection of the ER redox state, especially in living cells, is essential to understand the mechanism by which the redox state of the ER is maintained. However, methods to detect the redox state of the ER have not been well-established because of inefficient folding and stability of roGFP variants with oxidative redox potential like roGFP-iL. Here we have improved the folding efficiency of ER-targeted roGFP-iL (ERroGFP-iL) in cells by introducing superfolder GFP (sfGFP) mutations. Four specific amino acid substitutions (S30R, Y39N, T105N and I171V) greatly improved folding efficiency in Escherichia coli and in the ER of HeLa cells, as well as the thermostability of the purified proteins. Introduction of these mutations also enhanced the dynamic range for redox change both invitro and in the ER of living cells. ER-targeted roGFP-S4 (ERroGFP-S4) possessing these four mutations could detect physiological redox changes within the ER. ERroGFP-S4 is therefore a novel probe suitable for monitoring redox change in the ER. ERroGFP-S4 can be applied to detect aberrant ER redox states associated with various pathological conditions and to identify the mechanisms used to maintain the redox state of the ER.

The Intracellular Cargo Receptor ERGIC-53 Is Required for the Production of Infectious Arenavirus, Coronavirus, and Filovirus Particles

SummaryArenaviruses and hantaviruses cause severe human disease. Little is known regarding host proteins required for their propagation. We identified human proteins that interact with the glycoproteins (GPs) of a prototypic arenavirus and hantavirus and show that the lectin endoplasmic reticulum (ER)-Golgi intermediate compartment 53 kDa protein (ERGIC-53), a cargo receptor required for glycoprotein trafficking within the early exocytic pathway, associates with arenavirus, hantavirus, coronavirus, orthomyxovirus, and filovirus GPs. ERGIC-53 binds to arenavirus GPs through a lectin-independent mechanism, traffics to arenavirus budding sites, and is incorporated into virions. ERGIC-53 is required for arenavirus, coronavirus, and filovirus propagation; in its absence, GP-containing virus particles form but are noninfectious, due in part to their inability to attach to host cells. Thus, we have identified a class of pathogen-derived ERGIC-53 ligands, a lectin-independent basis for their association with ERGIC-53, and a role for ERGIC-53 in the propagation of several highly pathogenic RNA virus families.

Amino Acid Residues Critical for Endoplasmic Reticulum Export and Trafficking of Platelet-activating Factor Receptor

Several residues are conserved in the transmembrane domains (TMs) of G-protein coupled receptors. Here we demonstrate that a conserved proline, Pro(247), in TM6 of platelet-activating factor receptor (PAFR) is required for endoplasmic reticulum (ER) export and trafficking after agonist-induced internalization. Alanine-substituted mutants of the conserved residues of PAFRs, including P247A, were retained in the ER. Because a PAFR antagonist, Y-24180, acted as a pharmacological chaperone to rescue ER retention, this retention is due to misfolding of PAFR. Methylcarbamyl (mc)-PAF, a PAFR agonist, did not increase the cell surface expression of P247A, even though another ER-retained mutant, D63A, was effectively trafficked. Signaling and accumulation of the receptors in the early endosomes were observed in the mc-PAF-treated P247A-expressing cells, suggesting that P247A was trafficked to the cell surface by mc-PAF, and thereafter disappeared from the surface due to aberrant trafficking, e.g. enhanced internalization, deficiency in recycling, and/or accelerated degradation. The aberrant trafficking was confirmed with a sortase-A-mediated method for labeling cell surface proteins. These results demonstrate that the conserved proline in TM6 is crucial for intracellular trafficking of PAFR.

Molecular diagnosis of oculocutaneous albinism: new mutations in the OCA1–4 genes and practical aspects

Molecular diagnosis of oculocutaneous albinism: new mutations in the OCA1–4 genes and practical aspects

Mitochondria, endoplasmic reticulum and actin filament behavior after PDT with chloroaluminum phthalocyanine liposomal in HeLa cells

Photodynamic therapy (PDT) for cancer is a therapeutic modality in the treatment of tumors in which visible light is used to activate a photosensitizer. Cell membranes have been identified as an important intracellular target for singlet oxygen produced during the photochemical pathway. This study analyzed the cytotoxicity in specific cellular targets of a photosensitizer used in PDT in vitro. The photosensitizing effects of chloroaluminum phthalocyanine liposomal were studied on the mitochondria, cytoskeleton and endoplasmic reticulum of HeLa cells. Cells were irradiated with a diode laser working at 670 nm, energy density of 4.5 J/cm2 and power density of 45 mW/cm2. Fluorescence microscopic analysis of the mitochondria showed changes in membrane potential. After PDT treatment, the cytoskeleton and endoplasmic reticulum presented basic alterations in distribution. The combined effect of AlPHCl liposomal and red light in the HeLa cell line induced photodamage to the mitochondria, endoplasmic reticulum and actin filaments in the cytoskeleton.

Low temperature (15°C) induces COPII dissociation from membranes and slow exit from the endoplasmic reticulum in HeLa cells

Low temperature induces a transport blockade at the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) in cultured cells. Our previous studies support that the primary effect of low temperature is the detachment of COPI complexes from membranes. In the present study, we have used immunofluorescence and cryoimmunoelectron microscopy to investigate the effects of low temperature on both COPII and clathrin coat complexes in HeLa cells. Strikingly, COPII proteins moved from membranes to the cytosol at 15 degrees C, accumulating into electron-dense areas. In agreement with this observation, we also showed that ER exit is delayed in cells cultured at this temperature. In contrast, clathrin coat is not affected. Together, our results demonstrate that low temperature induces COPII dissociation from membranes and slow exit from the endoplasmic reticulum.

Novel Robinow syndrome causing mutations in the proximal region of the frizzled-like domain of ROR2 are retained in the endoplasmic reticulum

ROR2 is a member of the cell surface receptor tyrosine kinase (RTKs) family of proteins and is involved in the developmental morphogenesis of the skeletal, cardiovascular and genital systems. Mutations in ROR2 have been shown to cause two distinct human disorders, autosomal recessive Robinow syndrome and dominantly inherited Brachydactyly type B. The recessive form of Robinow syndrome is a disorder caused by loss-of-function mutations whereas Brachydactyly type B is a dominant disease and is presumably caused by gain-of-function mutations in the same gene. We have previously established that all the missense mutations causing Robinow syndrome in ROR2 are retained in the endoplasmic reticulum and therefore concluded that their loss of function is due to a defect in their intracellular trafficking. These mutations were in the distal portion of the frizzled-like cysteine rich domain and kringle domain. Here we report the identification of two novel mutations in the frizzled-like cysteine-rich domain of ROR2 causing Robinow syndrome. We establish the retention of the mutated proteins in the endoplasmic reticulum of HeLa cells and therefore failure to reach the plasma membrane. The clustering of Robinow-causing mutations in the extracellular frizzled-like cysteine-rich domain of ROR2 suggests a stringent requirement for the correct folding of this domain prior to export of ROR2 from the endoplasmic reticulum to the plasma membrane.

FGF Receptor Modulator Implicated in Brain Development

During brain development, instructive signals between the midbrain and the isthmus (the boundary region between the midbrain and hindbrain) control the development of the future tectum and cerebellum. One critical signal is mediated by fibroblast growth factor 8 (FGF8). Hirate and Okamoto report studies of this developmental regulatory system and identify a previously unrecognized regulator of FGF signaling. The authors previously identified a zebrafish gene they named canopy1 in a screen for genes expressed at the isthmus in response to signaling between the midbrain and the isthmus. Their new work showed that implantation of FGF8b-soaked beads in the midbrain of zebrafish embryos increased expression of canopy1 . Experiments in which expression of Canopy1 protein was blocked with antisense morpholino oligonucleotides showed that Canopy1 was essential for normal development of the tectum and cerebellum. Such inhibition of Canopy1 function also inhibited FGF8b-induced expression of a target gene, eng2a . The defect in FGF signaling appeared to be at the level of the receptor, because an artificially constructed FGF receptor activated by a dimerizing agent could still activate expression of eng2a in the absence of Canopy1. Canopy1 contains a sequence similar to ER (endoplasmic reticulum) retention signals, and, in transfected HeLa cells, this domain appeared to be required for retention of the protein in the ER. The human ortholog of Canopy1 was also localized to the ER in HeLa cells. Immunoprecipitation studies on extracts of cells transfected with a tagged Canopy1 protein along with full-length or fragmented FGF receptor protein suggested that Canopy1 may interact with the extracellular domain of the FGF receptor. The authors note that Canopy1 has structural similarity to saposins, proteins that bind sphingolipids, and speculate that Canopy1 could influence association of the receptor with lipid rafts. Y. Hirate, H. Okamoto, Canopy1, a novel regulator of FGF signaling around the midbrain-hindbrain boundary in zebrafish. Curr. Biol. 16 , 421-427 (2006). [PubMed]

Subplasmalemmal Mitochondria Modulate the Activity of Plasma Membrane Ca2+-ATPases

Mitochondria are dynamic organelles that modulate cellular Ca2+ signals by interacting with Ca2+ transporters on the plasma membrane or the endoplasmic reticulum (ER). To study how mitochondria dynamics affects cell Ca2+ homeostasis, we overexpressed two mitochondrial fission proteins, hFis1 and Drp1, and measured Ca2+ changes within the cytosol and the ER in HeLa cells. Both proteins fragmented mitochondria, decreased their total volume by 25-40%, and reduced the fraction of subplasmalemmal mitochondria by 4-fold. The cytosolic Ca2+ signals elicited by histamine were unaltered in cells lacking subplasmalemmal mitochondria as long as Ca2+ was present in the medium, but the signals were significantly blunted when Ca2+ was removed. Upon Ca2+ withdrawal, the free ER Ca2+ concentration decreased rapidly, and hFis1 cells were unable to respond to repetitive histamine stimulations. The loss of stored Ca2+ was due to an increased activity of plasma membrane Ca2+-ATPase (PMCA) pumps and was associated with an increased influx of Ca2+ and Mn2+ across store-operated Ca2+ channels. The increased Ca2+ influx compensated for the loss of stored Ca2+, and brief Ca2+ additions between successive agonist stimulations fully corrected subsequent histamine responses. We propose that the lack of subplasmalemmal mitochondria disrupts the transfer of Ca2+ from plasma membrane channels to the ER and that the resulting increase in subplasmalemmal [Ca2+] up-regulates the activity of PMCA. The increased Ca2+ extrusion promotes ER depletion and the subsequent activation of store-operated Ca2+ channels. Cells thus adapt to the lack of subplasmalemmal mitochondria by relying on external rather than on internal Ca2+ for signaling.

PAM mediates sustained inhibition of cAMP signaling by sphingosine-1-phosphate.

PAM (Protein Associated with Myc) is an almost ubiquitously expressed protein that is one of the most potent inhibitors of adenylyl cyclase activity known so far. Here we show that PAM is localized at the endoplasmic reticulum in HeLa cells and that upon serum treatment PAM is recruited to the plasma membrane, causing an inhibition of adenylyl cyclase activity. We purified the serum factor that induced PAM translocation and identified it as sphingosine-1-phosphate (S1P). Within 15 min after incubation with S1P, PAM appeared at the plasma membrane and was detectable for up to 120 min. Sphingosine-1-phosphate induced adenylyl cyclase inhibition in two phases: an initial (1-10 min) and a late (20-240 min) phase. The initial adenylyl cyclase inhibition was Gi-mediated and PAM independent. In the late phase, adenylyl cyclase inhibition was PAM dependent and attenuated cyclic AMP (cAMP) signaling by various cAMP-elevating signals. This makes PAM the longest lasting nontranscriptional regulator of adenylyl cyclase activity known to date and presents a novel mechanism for the temporal regulation of cAMP signaling.

Calcium release from the Golgi apparatus and the endoplasmic reticulum in HeLa cells stably expressing targeted aequorin to these compartments

Extracellular agonists mobilize Ca 2+ from SERCA-comprising intracellular Ca 2+ stores located in both the Golgi apparatus and the endoplasmic reticulum. Ca 2+ release from both these compartments was studied in HeLa cells stably expressing the luminescent Ca 2+ indicator aequorin specifically targeted to these compartments. Changes in lumenal [Ca 2+] as detected by the aequorin measurements were correlated with parallel changes in total Ca 2+ content of the stores. The latencies and initial rates of Ca 2+ release from the Golgi apparatus and the endoplasmic reticulum were quite similar. However, maximal Ca 2+ release measured with Golgi-targeted aequorin terminated faster than that from the endoplasmic reticulum. The rate and extent of Ca 2+ depletion from both compartments correlated well with the peak amplitude of the cytosolic [Ca 2+] rise. Time-course experiments further revealed that the peak of the cytosolic Ca 2+ response occurred before the lumenal [Ca 2+] reached its lowest level. We conclude that both the Golgi apparatus and the endoplasmic reticulum contribute to the rise in cytosolic [Ca 2+] upon agonist stimulation, but the kinetics of the Ca 2+ release are different.

Analysis of mitochondria, endoplasmic reticulum and actin filaments after PDT with AlPcS(4).

Photodynamic therapy (PDT) is a therapeutic modality for the treatment of tumors. This technique uses a visible light to activate a photosensitizer compounds, leading to a photo-oxidation process of biological tissue that can induce apoptosis or necrosis both in vivo and in vitro. However many of the cytotoxic effects remain an open question to be investigated. The cytotoxicity to specific cellular targets of classical photosensitizers used in the PDT in vitro has been analyzed in this work. The photosensitizing effects of Chloroaluminum Phthalocyanine Tetrasulfonate (AlPcS(4)) were studied on the mitochondria, cytoskeleton and endoplasmic reticulum of HeLa cells. The cells were irradiated with a diode laser (working at 670 nm; energy density of 4.5 J/cm(2 )and power density of 45 mW/cm(2)). The spectrofluorimetric analysis of the mitochondria showed changes in membrane potential. Cytoskeleton and endoplasmic reticulum showed basic alterations in distribution after PDT treatment, as an indicator of cellular death process.

Apoaequorin Monitors Degradation of Endoplasmic Reticulum (ER) Proteins Initiated by Loss of ER Ca2+

Apoaequorin was targeted to the cytosol, nucleus, and endoplasmic reticulum of HeLa cells in order to determine the effect of Ca2+ release from the ER on protein degradation. In resting cells apoaequorin had a rapid half-life (ca. 20–30 min) in the cytosol or nucleus, but was relatively stable for up to 24 h in the ER (t1/2 > 24 h). However, release of Ca2+ from the ER, initiated by the addition of inhibitors of the ER Ca2+/Mg2+ ATPase such as 2 μM thapsigargin or 1 μM ionomycin, initiated rapid loss of apoaequorin in the ER, but had no detectable effect on apoaequorin turnover in the cytosol nor the nucleus. This loss of apoprotein was not the result of secretion into the external fluid, and could not be inhibited by inhibitors of protein degradation by proteosomes. Proteolysis of apoaequorin in cell extracts (t1/2 < 20 min) was completely inhibited in the presence of 1 mM Ca2+, and this effect was independent of the ER retention signal KDEL at the C-terminus. Proteolysis was unaffected by the presence of selected serine protease inhibitors, or 10 μM Zn2+, a known caspase-3 inhibitor. The results show that apoaequorin can monitor proteolysis of ER proteins activated by loss of ER Ca2+. Several Ca2+-binding proteins exist in the ER, acting as the Ca2+ store and chaperones. Our results have important implications both for the role of ER Ca2+ in cell activation and stress and when using aequorin for monitoring free ER Ca2+ over long time periods.

Mistargeting of the lectin ERGIC-53 to the endoplasmic reticulum of HeLa cells impairs the secretion of a lysosomal enzyme.

ERGIC-53, a homo-oligomeric recycling protein associated with the ER-Golgi intermediate compartment (ERGIC), has properties of a mannose-selective lectin in vitro, suggesting that it may function as a transport receptor for glycoproteins in the early secretory pathway. To investigate if ERGIC-53 is involved in glycoprotein secretion, a mutant form of this protein was generated that is incapable of leaving the ER. If expressed in HeLa cells in a tetracycline-inducible manner, this mutant accumulated in the ER and retained the endogenous ERGIC-53 in this compartment, thus preventing its recycling. Mistargeting of ERGIC-53 to the ER did not alter the gross morphology of the early secretory pathway, including the distribution of beta'-COP. However, it impaired the secretion of one major glycoprotein, identified as the precursor of the lysosomal enzyme cathepsin C, while overexpression of wild-type ERGIC-53 had no effect on glycoprotein secretion. Transport of two other lysosomal enzymes and three post-Golgi membrane glycoproteins was unaffected by inactivating the recycling of ERGIC-53. The results suggest that the recycling of ERGIC-53 is required for efficient intracellular transport of a small subset of glycoproteins, but it does not appear to be essential for the majority of glycoproteins.