Diffusion in the endoplasmic reticulum of an aquaporin-2 mutant causing human nephrogenic diabetes insipidus

Mutations in the aquaporin-2 (AQP2) water channel cause the hereditary renal disease nephrogenic diabetes insipidus (NDI). The missense mutation AQP2-T126M causes human recessive NDI by retention at the endoplasmic reticulum (ER) of renal epithelial cells. To determine whether the ER retention of AQP2-T126M is due to relative immobilization in the ER, we measured by fluorescence recovery after photobleaching the intramembrane mobility of green fluorescent protein (GFP) chimeras containing human wild-type and mutant AQP2. In transfected LLC-PK1 renal epithelial cells, GFP-labeled AQP2-T126M was localized to the ER, and wild-type AQP2 to endosomes and the plasma membrane; both were localized to the ER after brefeldin A treatment. Photobleaching with image detection indicated that the GFP-AQP2 chimeras were freely mobile throughout the ER. Quantitative spot photobleaching revealed a diffusion-dependent irreversible process whose recovery depended on spot size and was abolished by paraformaldehyde fixation. In addition, a novel slow reversible fluorescence recovery (t(12) approximately 2 s) was characterized whose recovery was independent of spot size and not affected by fixation. AQP2 translational diffusion in the ER was not slowed by the T126M mutation; diffusion coefficients were (in cm(2)/s x 10(-)10) 2.6 +/- 0.5 (wild-type) and 3.0 +/- 0.4 (T126M). Much faster diffusion was found for a lipid probe (diOC(4)(3), 2.7 x 10(-)8 cm(2)/s) in the ER membrane and for unconjugated GFP in the aqueous ER lumen (6 x 10(-)8 cm(2)/s). ER diffusion of GFP-T126M was not significantly affected by up-regulation of molecular chaperones, cAMP activation, or actin filament disruption. ATP depletion by 2-deoxyglucose and azide resulted in comparable slowing/immobilization of wild-type and T126M AQP2. These results indicate that the ER retention of AQP2-T126M does not result from restricted or slowed mobility and suggest that the majority of AQP2-T126M is not aggregated or bound to slowly moving membrane proteins.     

Evidence for stabilization of aquaporin-2 folding mutants by N-linked glycosylation in endoplasmic reticulum

Aquaporin-2 (AQP2) is the vasopressin-sensitive water channel that regulates water reabsorption in the distal nephron collecting duct. Inherited AQP2 mutations that disrupt folding lead to nephrogenic diabetes insipidus (NDI) by targeting newly synthesized protein for degradation in the endoplasmic reticulum (ER). During synthesis, a subset of wild-type (WT) AQP2 is covalently modified by N-linked glycosylation at residue Asn123. To investigate the affect of glycosylation, we expressed WT AQP2 and four NDI-related mutants in Xenopus laevis oocytes and compared stability of glycosylated and nonglycosylated isoforms. In all constructs, 15–20% of newly synthesized AQP2 was covalently modified by N-linked glycosylation. At steady state, however, core glycosylated WT protein was nearly undetectable, whereas all mutants were found predominantly in the glycosylated form (60–70%). Pulse-chase metabolic labeling studies revealed that glycosylated isoforms of mutant AQP2 were significantly more stable than their nonglycosylated counterparts. For nonglycosylated isoforms, the half-life of WT AQP2 was significantly greater (>48 h) than that of mutant AQP2 (T126M 4.1 ± 1.0 h, A147T 4.2 ± 0.60 h, C181W 4.5 ± 0.50 h, R187C 6.8 ± 1.2 h). This is consistent with rapid turnover in the ER as previously reported. In contrast, the half-lives of mutant proteins containing N-linked glycans were similar to WT (25 h), indicating that differences in steady-state glycosylation profiles are caused by increased stability of glycosylated mutant proteins. These results suggest that addition of a single N-linked oligosaccharide moiety can partially compensate for ER folding defects induced by disease-related mutations. endoplasmic reticulum-associated degradation; nephrogenic diabetes insipidus; oocytes     

An impaired routing of wild-type aquaporin-2 after tetramerization with an aquaporin-2 mutant explains dominant nephrogenic diabetes insipidus

Autosomal recessive and dominant nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to vasopressin, are caused by mutations in the aquaporin-2 (AQP2) gene. Missense AQP2 proteins in recessive NDI have been shown to be retarded in the endoplasmic reticulum, whereas AQP2-E258K, an AQP2 mutant in dominant NDI, was retained in the Golgi complex. In this study, we identified the molecular mechanisms underlying recessive and dominant NDI. Sucrose gradient centrifugation of rat and human kidney proteins and subsequent immunoblotting revealed that AQP2 forms homotetramers. When expressed in oocytes, wild-type AQP2 and AQP2-E258K also formed homotetramers, whereas AQP2-R187C, a mutant in recessive NDI, was expressed as a monomer. Upon co-injection, AQP2-E258K, but not AQP2-R187C, was able to heterotetramerize with wild-type AQP2. Since an AQP monomer is the functional unit and AQP2-E258K is a functional but misrouted water channel, heterotetramerization of AQP2-E258K with wild-type AQP2 and inhibition of further routing of this complex to the plasma membrane is the cause of dominant NDI. This case of NDI is the first example of a dominant disease in which the 'loss-of-function' phenotype is caused by an impaired routing rather than impaired function of the wild-type protein.     

Efficient degradation of misfolded mutant Pma1 by endoplasmic reticulum-associated degradation requires Atg19 and the Cvt/autophagy pathway

Misfolded proteins are usually arrested in the endoplasmic reticulum (ER) and degraded by the ER-associated degradation (ERAD) machinery. Several mutant alleles of PMA1, the gene coding for the plasma membrane H(+)-ATPase, render misfolded proteins that are retained in the ER and degraded by ERAD. A subset of misfolded PMA1 mutants exhibit a dominant negative effect on yeast growth since, when coexpressed with the wild-type allele, both proteins are retained in the ER. We have used a pma1-D378T dominant negative mutant to identify new genes involved in ERAD. A genetic screen was performed for isolation of multicopy suppressors of a GAL1-pma1-D378T allele. ATG19, a member of the cytoplasm to vacuole targeting (Cvt) pathway, was found to suppress the growth arrest phenotype caused by the expression of pma1-D378T. ATG19 accelerates the degradation of pma1-D378T thus allowing the co-retained wild-type Pma1 to reach the plasma membrane. ATG19 was also able to suppress other dominant lethal PMA1 mutations. The degradation of the mutant ATPase occurs in the proteasome and requires intact both ERAD and Cvt/autophagy pathways. We propose the cooperation of both pathways for an efficient degradation of misfolded Pma1.     

Detection of aquaporin-2 in the plasma membranes of oocytes: a novel isolation method with improved yield and purity

Aquaporin-2 (AQP2) water channel mutations cause autosomal recessive and dominant nephrogenic diabetes insipidus (NDI). Expressed in oocytes, a mutant in dominant (AQP2-E258K), but not in recessive (AQP2-R187C), NDI conferred a specific dominant-negative effect on wild-type (wt) AQP2 water permeability (Pf) only at low expression levels. Since at these levels, the yield of conventional-isolated plasma membranes was too low, an improved technique to semiquantify AQP2 in the plasma membrane was needed. Antibodies against the C-loop of AQP2 were not applicable since they were unspecific and introduction of a tag into this loop caused misfolding and ER retardation. Membrane-impermeable biotin analogues turned out to label intracellular AQP2 proteins. Therefore, a method has been developed which generates a high yield of nearly pure plasma membranes, which enables semiquantification of plasma membrane proteins expressed at low levels in oocytes. Our new method allows for phenotype-genotype correlation studies in a wide range of channelopathies.     

Cell Surface Rescue of Kidney Anion Exchanger 1 Mutants by Disruption of Chaperone Interactions

Mutations in the human kidney anion exchanger 1 (kAE1) membrane glycoprotein cause impaired urine acidification resulting in distal renal tubular acidosis (dRTA). Dominant and recessive dRTA kAE1 mutants exhibit distinct trafficking defects with retention in the endoplasmic reticulum (ER), Golgi, or mislocalization to the apical membrane in polarized epithelial cells. We examined the interaction of kAE1 with the quality control system responsible for the folding of membrane glycoproteins and the retention and degradation of misfolded mutants. Using small molecule inhibitors to disrupt chaperone interactions, two functional, dominant kAE1 mutants (R589H and R901stop), retained in the ER and targeted to the proteasome for degradation by ubiquitination, were rescued to the basolateral membrane of Madin-Darby canine kidney cells. In contrast, the Golgi-localized, recessive G701D and the severely misfolded, ER-retained dominant Southeast Asian ovalocytosis (SAO) mutants were not rescued. These results show that functional dRTA mutants are retained in the ER due to their interaction with molecular chaperones, particularly calnexin, and that disruption of these interactions can promote their escape from the ER and cell surface rescue.     

Depletion of Molecular Chaperones from the Endoplasmic Reticulum and Fragmentation of the Golgi Apparatus Associated with Pathogenesis in Pelizaeus-Merzbacher Disease

Missense mutations in the proteolipid protein 1 (PLP1) gene cause a wide spectrum of hypomyelinating disorders, from mild spastic paraplegia type 2 to severe Pelizaeus-Merzbacher disease (PMD). Mutant PLP1 accumulates in the endoplasmic reticulum (ER) and induces ER stress. However, the link between the clinical severity of PMD and the cellular response induced by mutant PLP1 remains largely unknown. Accumulation of misfolded proteins in the ER generally leads to up-regulation of ER chaperones to alleviate ER stress. Here, we found that expression of the PLP1-A243V mutant, which causes severe disease, depletes some ER chaperones with a KDEL (Lys-Asp-Glu-Leu) motif, in HeLa cells, MO3.13 oligodendrocytic cells, and primary oligodendrocytes. The same PLP1 mutant also induces fragmentation of the Golgi apparatus (GA). These organelle changes are less prominent in cells with milder disease-associated PLP1 mutants. Similar changes are also observed in cells expressing another disease-causing gene that triggers ER stress, as well as in cells treated with brefeldin A, which induces ER stress and GA fragmentation by inhibiting GA to ER trafficking. We also found that mutant PLP1 disturbs localization of the KDEL receptor, which transports the chaperones with the KDEL motif from the GA to the ER. These data show that PLP1 mutants inhibit GA to ER trafficking, which reduces the supply of ER chaperones and induces GA fragmentation. We propose that depletion of ER chaperones and GA fragmentation induced by mutant misfolded proteins contribute to the pathogenesis of inherited ER stress-related diseases and affect the disease severity.     

Correctors promote folding of the CFTR in the endoplasmic reticulum

Cystic fibrosis (CF) is most commonly caused by deletion of a residue (DeltaF508) in the CFTR (cystic fibrosis transmembrane conductance regulator) protein. The misfolded mutant protein is retained in the ER (endoplasmic reticulum) and is not trafficked to the cell surface (misprocessed mutant). Corrector molecules such as corr-2b or corr-4a are small molecules that increase the amount of functional CFTR at the cell surface. Correctors may function by stabilizing CFTR at the cell surface or by promoting folding in the ER. To test whether correctors promoted folding of CFTR in the ER, we constructed double-cysteine CFTR mutants that would be retained in the ER and only undergo cross-linking when the protein folds into a native structure. The mature form, but not the immature forms, of M348C(TM6)/T1142C(TM12) (where TM is transmembrane segment), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C(TM12) mutants were efficiently cross-linked. Mutations to the COPII (coatamer protein II) exit motif (Y(563)KDAD(567)) were then made in the cross-linkable cysteine mutants to prevent the mutant proteins from leaving the ER. Membranes were prepared from the mutants expressed in the absence or presence of correctors and subjected to disulfide cross-linking analysis. The presence of correctors promoted folding of the mutants as the efficiency of cross-linking increased from approx. 2-5% to 22-35%. The results suggest that correctors interact with CFTR in the ER to promote folding of the protein into a native structure.     

Endoplasmic Reticulum Oxidoreductin-1α (Ero1α) Improves Folding and Secretion of Mutant Proinsulin and Limits Mutant Proinsulin-induced Endoplasmic Reticulum Stress

Upon chronic up-regulation of proinsulin synthesis, misfolded proinsulin can accumulate in the endoplasmic reticulum (ER) of pancreatic β-cells, promoting ER stress and type 2 diabetes mellitus. In Mutant Ins-gene-induced Diabetes of Youth (MIDY), misfolded mutant proinsulin impairs ER exit of co-expressed wild-type proinsulin, limiting insulin production and leading to eventual β-cell death. In this study we have investigated the hypothesis that increased expression of ER oxidoreductin-1α (Ero1α), despite its established role in the generation of H₂O₂, might nevertheless be beneficial in limiting proinsulin misfolding and its adverse downstream consequences. Increased Ero1α expression is effective in promoting wild-type proinsulin export from cells co-expressing misfolded mutant proinsulin. In addition, we find that upon increased Ero1α expression, some of the MIDY mutants themselves are directly rescued from ER retention. Secretory rescue of proinsulin-G(B23)V is correlated with improved oxidative folding of mutant proinsulin. Indeed, using three different variants of Ero1α, we find that expression of either wild-type or an Ero1α variant lacking regulatory disulfides can rescue mutant proinsulin-G(B23)V, in parallel with its ability to provide an oxidizing environment in the ER lumen, whereas beneficial effects were less apparent for a redox-inactive form of Ero1. Increased expression of protein disulfide isomerase antagonizes the rescue provided by oxidatively active Ero1. Importantly, ER stress induced by misfolded proinsulin was limited by increased expression of Ero1α, suggesting that enhancing the oxidative folding of proinsulin may be a viable therapeutic strategy in the treatment of type 2 diabetes.     

Intracellular transport, assembly, and degradation of wild-type and disease-linked mutant gap junction proteins

More than 130 different mutations in the gap junction integral plasma membrane protein connexin32 (Cx32) have been linked to the human peripheral neuropathy X-linked Charcot-Marie-Tooth disease (CMTX). How these various mutants are processed by the cell and the mechanism(s) by which they cause CMTX are unknown. To address these issues, we have studied the intracellular transport, assembly, and degradation of three CMTX-linked Cx32 mutants stably expressed in PC12 cells. Each mutant had a distinct fate: E208K Cx32 appeared to be retained in the endoplasmic reticulum (ER), whereas both the E186K and R142W mutants were transported to perinuclear compartments from which they trafficked either to lysosomes (R142W Cx32) or back to the ER (E186K Cx32). Despite these differences, each mutant was soluble in nonionic detergent but unable to assemble into homomeric connexons. Degradation of both mutant and wild-type connexins was rapid (t(1/2) < 3 h) and took place at least in part in the ER by a process sensitive to proteasome inhibitors. The mutants studied are therefore unlikely to cause disease by accumulating in degradation-resistant aggregates but instead are efficiently cleared from the cell by quality control processes that prevent abnormal connexin molecules from traversing the secretory pathway.     

TRAM1 is involved in disposal of ER membrane degradation substrates

ER quality control consists of monitoring protein folding and targeting misfolded proteins for proteasomal degradation. ER stress results in an unfolded protein response (UPR) that selectively upregulates proteins involved in protein degradation, ER expansion, and protein folding. Given the efficiency in which misfolded proteins are degraded, there likely exist cellular factors that enhance the export of proteins across the ER membrane. We have reported that translocating chain-associated membrane protein 1 (TRAM1), an ER-resident membrane protein, participates in HCMV US2- and US11-mediated dislocation of MHC class I heavy chains (Oresic, K., Ng, C.L., and Tortorella, D. 2009). Consistent with the hypothesis that TRAM1 is involved in the disposal of misfolded ER proteins, cells lacking TRAM1 experienced a heightened UPR upon acute ER stress, as evidenced by increased activation of unfolded protein response elements (UPRE) and elevated levels of NF-κB activity. We have also extended the involvement of TRAM1 in the selective degradation of misfolded ER membrane proteins Cln6^M241T and US2, but not the soluble degradation substrate α₁-antitrypsin null_HK. These degradation model systems support the paradigm that TRAM1 is a selective factor that can enhance the dislocation of ER membrane proteins.     

Chaperone-mediated hierarchical control in targeting misfolded proteins to aggresomes

Protein misfolding is a common event in living cells. Molecular chaperones not only assist protein folding; they also facilitate the degradation of misfolded polypeptides. When the intracellular degradative capacity is exceeded, juxtanuclear aggresomes are formed to sequester misfolded proteins. Despite the well-established role of chaperones in both protein folding and degradation, how chaperones regulate the aggregation process remains controversial. Here we investigate the molecular mechanisms underlying aggresome formation in mammalian cells. Analysis of the chaperone requirements for the fate of misfolded proteins reveals an unexpected role of heat shock protein 70 (Hsp70) in promoting aggresome formation. This proaggregation function of Hsp70 relies on the interaction with the cochaperone ubiquitin ligase carboxyl terminal of Hsp70/Hsp90 interacting protein (CHIP). Disrupting Hsp70-CHIP interaction prevents the aggresome formation, whereas a dominant-negative CHIP mutant sensitizes the aggregation of misfolded protein. This accelerated aggresome formation also relies on the stress-induced cochaperone Bcl2-associated athanogene 3. Our results indicate that a hierarchy of cochaperone interaction controls different aspects of the intracellular protein triage decision, extending the function of Hsp70 from folding and degradation to aggregation.     

ERAD substrates: Which way out?

Global folding of polypeptides entering the endoplasmic reticulum (ER) starts as soon as they emerge from the narrow Sec61 translocon. Attainment of the native structure can take from several minutes to hours, depending on the gene product. Until then, non-native folding intermediates must be protected from molecular chaperones that recognize misfolded determinants and could prematurely interrupt folding programs by re-directing them to disposal pathways. On the other hand, futile folding attempts must actively be stopped to prevent intraluminal accumulation of defective cargo. This review describes recent advances in understanding how terminally misfolded polypeptides are extracted from the folding environment and directed to specific dislocons within the ER membrane for transfer to the cytoplasm for proteasome-mediated degradation.     

A Dominant Negative Mutant of Pma1 Interferes with the Folding of the Wild Type Enzyme

Misfolded proteins are usually arrested in the endoplasmic reticulum (ER) and degraded by the ER-associated degradation (ERAD) machinery. Several mutant alleles of PMA1 , the gene coding for the plasma membrane H ⁺-ATPase, render misfolded proteins that are subjected to ERAD. A subset of misfolded PMA1 mutants exhibits a dominant negative effect on yeast growth since, when co-expressed with the wild type allele, both proteins are retained in the ER and degraded. We have used a PMA1 -D378T dominant lethal allele to analyse the mechanism underlying the retention of the wild type enzyme by the dominant negative mutant. A genetic screen was performed for isolation of intragenic suppressors of PMA1 -D378T allele. This analysis pointed to transmembrane helix 10 (TM10) as an important element in the establishment of the dominant lethality. Deletion of the TM10 was able to suppress not only the PMA1 -D378T but all the dominant lethal alleles tested. Biochemical analyses suggest that dominant lethal proteins obstruct, through TM10, the correct folding of the wild type enzyme leading to its retention and degradation by ERAD.     

IRE1 directs proteasomal and lysosomal degradation of misfolded rhodopsin

Endoplasmic reticulum (ER) is responsible for folding of secreted and membrane proteins in eukaryotic cells. Disruption of ER protein folding leads to ER stress. Chronic ER stress can cause cell death and is proposed to underlie the pathogenesis of many human diseases. Inositol-requiring enzyme 1 (IRE1) directs a key unfolded protein response signaling pathway that controls the fidelity of ER protein folding. IRE1 signaling may be particularly helpful in preventing chronic ER stress and cell injury by alleviating protein misfolding in the ER. To examine this, we used a chemical-genetic approach to selectively activate IRE1 in mammalian cells and tested how artificial IRE1 signaling affected the fate of misfolded P23H rhodopsin linked to photoreceptor cell death. We found that IRE1 signaling robustly promoted the degradation of misfolded P23H rhodopsin without affecting its wild-type counterpart. We also found that IRE1 used both proteasomal and lysosomal degradation pathways to remove P23H rhodopsin. Surprisingly, when one degradation pathway was compromised, IRE1 signaling could still promote misfolded rhodopsin degradation using the remaining pathway. Last, we showed that IRE1 signaling also reduced levels of several other misfolded rhodopsins with lesser effects on misfolded cystic fibrosis transmembrane conductance regulator. Our findings reveal the diversity of proteolytic mechanisms used by IRE1 to eliminate misfolded rhodopsin.