Dominant and recessive distal renal tubular acidosis mutations of kidney anion exchanger 1 induce distinct trafficking defects in MDCK cells

Distal renal tubular acidosis (dRTA), a kidney disease resulting in defective urinary acidification, can be caused by dominant or recessive mutations in the kidney Cl-/HCO3- anion exchanger (kAE1), a glycoprotein expressed in the basolateral membrane of alpha-intercalated cells. We compared the effect of two dominant (R589H and S613F) and two recessive (S773P and G701D) dRTA point mutations on kAE1 trafficking in Madin-Darby canine kidney (MDCK) epithelial cells. In contrast to wild-type (WT) kAE1 that was localized to the basolateral membrane, the dominant mutants (kAE1 R589H and S613F) were retained in the endoplasmic reticulum (ER) in MDCK cells, with a few cells showing in addition some apical localization. The recessive mutant kAE1 S773P, while misfolded and largely retained in the ER in non-polarized MDCK cells, was targeted to the basolateral membrane after polarization. The other recessive mutants, kAE1 G701D and designed G701E, G701R but not G701A or G701L mutants, were localized to the Golgi in both non-polarized and polarized cells. The results suggest that introduction of a polar mutation into a transmembrane segment resulted in Golgi retention of the recessive G701D mutant. When coexpressed, the dominant mutants retained kAE1 WT intracellularly, while the recessive mutants did not. Coexpression of recessive G701D and S773P mutants in polarized cells showed that these proteins could interact, yet no G701D mutant was detected at the basolateral membrane. Therefore, compound heterozygous patients expressing both recessive mutants (G701D/S773P) likely developed dRTA due to the lack of a functional kAE1 at the basolateral surface of alpha-intercalated cells.     

Trafficking defects of a novel autosomal recessive distal renal tubular acidosis mutant (S773P) of the human kidney anion exchanger (kAE1)

Autosomal dominant and recessive distal renal tubular acidosis (dRTA) can be caused by mutations in the anion exchanger 1 (AE1 or SLC4A1) gene, which encodes the erythroid chloride/bicarbonate anion exchanger membrane glycoprotein (eAE1) and a truncated kidney isoform (kAE1). The biosynthesis and trafficking of kAE1 containing a novel recessive missense dRTA mutation (kAE1 S773P) was studied in transiently transfected HEK-293 cells, expressing the mutant alone or in combination with wild-type kAE1 or another recessive mutant, kAE1 G701D. The kAE1 S773P mutant was expressed at a three times lower level than wild-type, had a 2-fold decrease in its half-life, and was targeted for degradation by the proteasome. It could not be detected at the plasma membrane in human embryonic kidney cells and showed predominant endoplasmic reticulum immunolocalization in both human embryonic kidney and LLC-PK1 cells. The oligosaccharide on a kAE1 S773P N-glycosylation mutant (N555) was not processed to the complex form indicating impaired exit from the endoplasmic reticulum. The kAE1 S773P mutant showed decreased binding to an inhibitor affinity resin and increased sensitivity to proteases, suggesting that it was not properly folded. The other recessive dRTA mutant, kAE1 G701D, also exhibited defective trafficking to the plasma membrane. The recessive kAE1 mutants formed dimers like wild-type AE1 and could hetero-oligomerize with wild-type kAE1 or with each other. Hetero-oligomers of wild-type kAE1 with recessive kAE1 S773P or G701D, in contrast to the dominant kAE1 R589H mutant, were delivered to the plasma membrane.     

Dominant-negative effect of Southeast Asian ovalocytosis anion exchanger 1 in compound heterozygous distal renal tubular acidosis

The human chloride/bicarbonate AE1 (anion exchanger) is a dimeric glycoprotein expressed in the red blood cell membrane,and expressed as an N-terminal (Delta1-65) truncated form, kAE1(kidney AE1), in the basolateral membrane of alpha-intercalated cells in the distal nephron. Mutations in AE1 can cause SAO (Southeast Asian ovalocytosis) or dRTA (distal renal tubular acidosis), an inherited kidney disease resulting in impaired acid secretion. The dominant SAO mutation (Delta400-408) that results in an inactive transporter and altered erythrocyte shape occurs in manydRTA families, but does not itself result in dRTA. Compound heterozygotes of four dRTA mutations (R602H, G701D, DeltaV850 and A858D) with SAO exhibit dRTA and abnormal red blood cell properties. Co-expression of kAE1 and kAE1 SAO with the dRTAmutantswas studied in polarized epithelial MDCK(Madin-Darbycanine kidney) cells. Like SAO, the G701D and DeltaV850 mutants were predominantly retained intracellularly, whereas the R602H and A858D mutants could traffic to the basolateral membrane. When co-expressed in transfected cells, kAE1 WT (wild-type)and kAE1 SAO could interact with the dRTA mutants. MDCK cells co-expressing kAE1 SAO with kAE1 WT, kAE1 R602Hor kAE1 A858D showed a decrease in cell-surface expression of the co-expressed proteins. When co-expressed, kAE1 WT colocalized with the kAE1 R602H, kAE1 G701D, kAE1 DeltaV850 and kAE1 A858D mutants at the basolateral membrane, whereaskAE1 SAO co-localized with kAE1 WT, kAE1 R602H, kAE1 G701D, kAE1 DeltaV850 and kAE1 A858D in MDCK cells. The decrease in cell-surface expression of the dRTAmutants as a result of the interaction with kAE1 SAO would account for the impaired expression of functional kAE1 at the basolateral membrane of alpha-intercalated cells, resulting in dRTA in compound heterozygous patients.     

Functional Rescue of a Kidney Anion Exchanger 1 Trafficking Mutant in Renal Epithelial Cells

Mutations in the SLC4A1 gene encoding the anion exchanger 1 (AE1) can cause distal renal tubular acidosis (dRTA), a disease often due to mis-trafficking of the mutant protein. In this study, we investigated whether trafficking of a Golgi-retained dRTA mutant, G701D kAE1, or two dRTA mutants retained in the endoplasmic reticulum, C479W and R589H kAE1, could be functionally rescued to the plasma membrane of Madin-Darby Canine Kidney (MDCK) cells. Treatments with DMSO, glycerol, the corrector VX-809, or low temperature incubations restored the basolateral trafficking of G701D kAE1 mutant. These treatments had no significant rescuing effect on trafficking of the mis-folded C479W or R589H kAE1 mutants. DMSO was the only treatment that partially restored G701D kAE1 function in the plasma membrane of MDCK cells. Our experiments show that trafficking of intracellularly retained dRTA kAE1 mutants can be partially restored, and that one chemical treatment rescued both trafficking and function of a dRTA mutant. These studies provide an opportunity to develop alternative therapeutic solutions for dRTA patients.     

Defective kidney anion-exchanger 1 (AE1, Band 3) trafficking in dominant distal renal tubular acidosis (dRTA)

dRTA (distal renal tubular acidosis) results from the failure of the a-intercalated cells in the distal tubule of the nephron to acidify the urine. A truncated form of AE1 (anion-exchanger 1; Band 3), kAE1 (kidney isoform of AE1), is located in the basolateral membrane of the intercalated cell. Mutations in the AE1 gene cause autosomal dominant and recessive forms of dRTA. All the dominant dRTA mutations investigated cause aberrant trafficking of kAE1, resulting in its intracellular retention or mistargeting to the apical plasma membrane. Therefore the intracellular retention of hetero-oligomers containing wild-type and dRTA mutants, or the mistargeted protein in the apical membrane neutralizing acid secretion, explains dominant dRTA. The kAE1 (Arg(901)-->stop) mutant has been studied in more detail, since the mistargeting kAE1 (Arg(901)-->stop) from the basolateral to the apical membrane is consistent with the removal of a basolateral localization signal. The C-terminal amino acids deleted by the Arg(901)-->stop mutation, contain a tyrosine motif and a type II PDZ interaction domain. The tyrosine residue (Tyr(904)), but not the PDZ domain, is critical for basolateral localization. In the absence of the N-terminus of kAE1, the C-terminus was not sufficient to localize kAE1 to the basolateral membrane. This suggests that a determinant within the kAE1 N-terminus co-operates with the C-terminus for kAE1 basolateral localization. Interestingly, Tyr(359), in the N-terminal domain, and Tyr(904) in the C-terminus of AE1 are phosphorylated in red blood cells. A potential scheme is suggested where successive phosphorylation of these residues is necessary for correct localization and recycling of kAE1 to the basolateral membrane.     

Impaired trafficking and intracellular retention of mutant kidney anion exchanger 1 proteins (G701D and A858D) associated with distal renal tubular acidosis

Abstract

Novel compound heterozygous mutations, G701D, a recessive mutation, and A858D, a mild dominant mutation, of human solute carrier family 4, anion exchanger, member 1 (SLC4A1) were identified in two pediatric patients with distal renal tubular acidosis (dRTA). To examine the interaction, trafficking, and cellular localization of the wild-type and two mutant kidney AE1 (kAE1) proteins, we expressed the proteins alone or together in human embryonic kidney (HEK) 293T and Madin-Darby canine kidney (MDCK) epithelial cells. In individual expressions, wild-type kAE1 was localized at the cell surface of HEK 293T and the basolateral membrane of MDCK cells. In contrast, kAE1 G701D was mainly retained intracellularly, while kAE1 A858D was observed intracellularly and at the cell surface. In co-expression experiments, wild-type kAE1 formed heterodimers with kAE1 G701D and kAE1 A858D, and promoted the cell surface expression of the mutant proteins. The co-expressed kAE1 G701D and A858D could also form heterodimers but showed predominant intracellular retention in HEK 293T and MDCK cells. Thus impaired trafficking of the kAE1 G701D and A858D mutants would lead to a profound decrease in functional kAE1 at the basolateral membrane of α-intercalated cells in the distal nephron of the patients with dRTA.     

Adaptor protein 1 B mu subunit does not contribute to the recycling of kAE1 protein in polarized renal epithelial cells

Mutations in the gene encoding the kidney anion exchanger 1 (kAE1) can lead to distal renal tubular acidosis (dRTA). dRTA mutations reported within the carboxyl (C)-terminal tail of kAE1 result in apical mis-targeting of the exchanger in polarized renal epithelial cells. As kAE1 physically interacts with the μ subunit of epithelial adaptor protein 1 B (AP-1B), we investigated the role of heterologously expressed μ1B subunit of the AP-1B complex for kAE1 retention to the basolateral membrane in polarized porcine LLC-PK1 renal epithelial cells that are devoid of endogenous AP-1B. We confirmed the interaction and close proximity between kAE1 and μ1B using immunoprecipitation and proximity ligation assay, respectively. Expressing the human μ1B subunit in these cells decreased significantly the amount of cell surface kAE1 at the steady state, but had no significant effect on kAE1 recycling and endocytosis. We show that (i) heterologous expression of μ1B displaces the physical interaction of endogenous GAPDH with kAE1?WT supporting that both AP-1B and GAPDH proteins bind to an overlapping site on kAE1 and (ii) phosphorylation of tyrosine 904 within the potential YDEV interaction motif does not alter the kAE1/AP-1B interaction. We conclude that μ1B subunit is not involved in recycling of kAE1.     

A novel missense mutation in AE1 causing autosomal dominant distal renal tubular acidosis retains normal transport function but is mistargeted in polarized epithelial cells

Mutations in SLC4A1, encoding the chloride-bicarbonate exchanger AE1, cause distal renal tubular acidosis (dRTA), a disease of defective urinary acidification by the distal nephron. In this study we report a novel missense mutation, G609R, causing dominant dRTA in affected members of a large Caucasian pedigree who all exhibited metabolic acidosis with alkaline urine, prominent nephrocalcinosis, and progressive renal impairment. To investigate the potential disease mechanism, the consequent effects of this mutation were determined. We first assessed anion transport function of G609R by expression in Xenopus oocytes. Western blotting and immunofluorescence demonstrated that the mutant protein was expressed at the oocyte cell surface. Measuring chloride and bicarbonate fluxes revealed normal 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-inhibitable anion exchange, suggesting that loss-of-function of kAE1 cannot explain the severe disease phenotype in this kindred. We next expressed epitope-tagged wild-type or mutant kAE1 in Madin-Darby canine kidney cells. In monolayers grown to polarity, mutant kAE1 was detected subapically and at the apical membrane, as well as at the basolateral membrane, in contrast to the normal basolateral appearance of wild-type kAE1. These findings suggest that the seventh transmembrane domain that contains Gly-609 plays an important role in targeting kAE1 to the correct cell surface compartment. They confirm that dominant dRTA is associated with non-polarized trafficking of the protein, with no significant effect on anion transport function in vitro, which remains an unusual mechanism of human disease.     

Unraveling trafficking of the kidney anion exchanger 1 in polarized MDCK epithelial cells.

Kidney anion exchanger 1 (kAE1) is a membrane glycoprotein expressed at the basolateral membrane of type A intercalated cells in the kidney collecting tubule. Mutations occurring in the gene encoding this protein can give rise to distal renal tubular acidosis (dRTA), a disease characterized by an impaired urine acidification, nephrocalcinosis, and renal failure. Here we review how the study of dRTA mutants in polarized epithelial cells has shed light on the cellular mechanisms resulting in this renal disease.     

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.     

Human kanadaptin and kidney anion exchanger 1 (kAE1) do not interact in transfected HEK 293 cells

Kanadaptin (kidney anion exchanger adaptor protein) is a widely expressed protein, shown previously to interact with the cytosolic domain of mouse Cl-/HCO3- anion exchanger 1 (kAE1) but not erythroid AE1 (eAE1) by a yeast-two hybrid assay. Kanadaptin was co-localized with kAE1 in intracellular membranes but not at the plasma membrane in alpha-intercalated cells of rabbit kidney. It was suggested that kanadaptin is an adaptor protein or chaperone involved in targeting kAE1 to the plasma membrane. To test this hypothesis, the interaction of human kanadaptin with human kAE1 was studied in co-transfected HEK293 cells. Human kanadaptin contains 796 amino acids and was immuno-detected as a 90 kDa protein in transfected cells. Pulse-chase experiments showed that it has a half-life (t1/2) of 7 h. Human kanadaptin was localized predominantly to the nucleus, whereas kAE1 was present intracellularly and at the plasma membrane. Trafficking of kAE1 from its site of synthesis in the endoplasmic reticulum to the plasma membrane was unaffected by co-expression of human kanadaptin. Moreover, we found that no interaction between human kanadaptin and kAE1 or eAE1 could be detected in co-transfected cells either by co-immunoprecipitation or by histidine6-tagged co-purification. Taken together, we found that human kanadaptin did not interact with kAE1 and had no effect on trafficking of kAE1 to the plasma membrane in transfected cells. Kanadaptin may not be involved in the biosynthesis and targeting of kAE1. As such, defects in kanadaptin and its interaction with kAE1 are unlikely to be involved in the pathogenesis of the inherited kidney disease, distal renal tubular acidosis (dRTA).     

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.     

Cleavage of Notch1 by granzyme B disables its transcriptional activity

AE1 (anion exchanger 1) and protein 4.2 associate in a protein complex bridging the erythrocyte membrane and cytoskeleton; disruption of the complex results in unstable erythrocytes and HS (hereditary spherocytosis). Three HS mutations (E40K, G130R and P327R) in cdAE1 (the cytoplasmic domain of AE1) occur with deficiencies of protein 4.2. The interaction of wild-type AE1, AE1HS mutants, mdEA1 (the membrane domain of AE1), kAE1 (the kidney isoform of AE1) and AE1SAO (Southeast Asian ovalocytosis AE1) with protein 4.2 was examined in transfected HEK (human embryonic kidney)-293 cells. The HS mutants had wild-type expression levels and plasma-membrane localization. Protein 4.2 expression was not dependent on AE1. Protein 4.2 was localized throughout the cytoplasm and co-localized at the plasma membrane with the HS mutants mdAE1 and kAE1, but at the ER (endoplasmic reticulum) with AE1SAO. Pull-down assays revealed diminished levels of protein 4.2 associated with the HS mutants relative to AE1. The mdAE1 did not bind protein 4.2, whereas kAE1 and AE1SAO bound wild-type amounts of protein 4.2. A protein 4.2 fatty acylation mutant, G2A/C173A, had decreased plasma-membrane localization compared with wild-type protein 4.2, and co-expression with AE1 enhanced its plasma-membrane localization. Subcellular fractionation showed the majority of wild-type and G2A/C173A protein 4.2 was associated with the cytoskeleton of HEK-293 cells. The present study shows that cytoplasmic HS mutants cause impaired binding of protein 4.2 to AE1, leaving protein 4.2 susceptible to loss during erythrocyte development.     

Human kidney anion exchanger 1 interacts with kinesin family member 3B (KIF3B)

Impaired trafficking of human kidney anion exchanger 1 (kAE1) to the basolateral membrane of α-intercalated cells of the kidney collecting duct leads to the defect of the Cl⁻/ exchange and the failure of proton (H⁺) secretion at the apical membrane of these cells, causing distal renal tubular acidosis (dRTA). In the sorting process, kAE1 interacts with AP-1 mu1A, a subunit of AP-1A adaptor complex. However, it is not known whether kAE1 interacts with motor proteins in its trafficking process to the plasma membrane or not. We report here that kAE1 interacts with kinesin family member 3B (KIF3B) in kidney cells and a dileucine motif at the carboxyl terminus of kAE1 contributes to this interaction. We have also demonstrated that kAE1 co-localizes with KIF3B in human kidney tissues and the suppression of endogenous KIF3B in HEK293T cells by small interfering RNA (siRNA) decreases membrane localization of kAE1 but increases its intracellular accumulation. All results suggest that KIF3B is involved in the trafficking of kAE1 to the plasma membrane of human kidney α-intercalated cells.     

Adaptor protein 1 complexes regulate intracellular trafficking of the kidney anion exchanger 1 in epithelial cells

Distal renal tubular acidosis (dRTA) can be caused by mutations in the gene encoding the anion exchanger 1 (AE1) and is characterized by defective urinary acidification, metabolic acidosis, and renal stones. AE1 is expressed at the basolateral membrane of type A intercalated cells in the renal cortical collecting duct (kAE1). Two dRTA mutations result in the carboxyl-terminal truncation of kAE1; in one case, the protein trafficked in a nonpolarized way in epithelial cells. A recent yeast two-hybrid assay showed that the carboxyl-terminal cytosolic domain of AE1 interacts with adaptor protein complex 1 (AP-1A) subunit μ1A (mu-1A; Sawasdee N, Junking M, Ngaojanlar P, Sukomon N, Ungsupravate D, Limjindaporn T, Akkarapatumwong V, Noisakran S, Yenchitsomanus PT. Biochem Biophys Res Commun 401: 85-91, 2010). Here, we show the interaction between kAE1 and mu-1A and B in vitro by reciprocal coimmunoprecipitation in epithelial cells and in vivo by coimmunoprecipitation from mouse kidney extract. When endogenous mu-1A (and to a lesser extent mu-1B) was reduced, kAE1 protein was unable to traffic to the plasma membrane and was rapidly degraded via a lysosomal pathway. Expression of either small interfering RNA-resistant mu-1A or mu-1B stabilized kAE1 in these cells. We also show that newly synthesized kAE1 does not traffic through recycling endosomes to the plasma membrane, suggesting that AP-1B, located in recycling endosomes, is not primarily involved in trafficking of newly synthesized kAE1 when AP-1A is present in the cells. Our data demonstrate that AP-1A regulates processing of the basolateral, polytopic membrane protein kAE1 to the cell surface and that both AP-1A and B adaptor complexes are required for normal kAE1 trafficking.     

Human kanadaptin and kidney anion exchanger 1 (kAE1) do not interact in transfected HEK 293 cells

Kanadaptin (k¯idney anion exchanger adaptor protein) is a widely expressed protein, shown previously to interact with the cytosolic domain of mouse Cl^?/HCO₃^? anion exchanger 1 (kAE1) but not erythroid AE1 (eAE1) by a yeast-two hybrid assay. Kanadaptin was co-localized with kAE1 in intracellular membranes but not at the plasma membrane in α-intercalated cells of rabbit kidney. It was suggested that kanadaptin is an adaptor protein or chaperone involved in targeting kAE1 to the plasma membrane. To test this hypothesis, the interaction of human kanadaptin with human kAE1 was studied in co-transfected HEK293 cells. Human kanadaptin contains 796 amino acids and was immuno-detected as a 90 kDa protein in transfected cells. Pulse-chase experiments showed that it has a half-life (t_1/2) of 7 h. Human kanadaptin was localized predominantly to the nucleus, whereas kAE1 was present intracellularly and at the plasma membrane. Trafficking of kAE1 from its site of synthesis in the endoplasmic reticulum to the plasma membrane was unaffected by co-expression of human kanadaptin. Moreover, we found that no interaction between human kanadaptin and kAE1 or eAE1 could be detected in co-transfected cells either by co-immunoprecipitation or by histidine6-tagged co-purification. Taken together, we found that human kanadaptin did not interact with kAE1 and had no effect on trafficking of kAE1 to the plasma membrane in transfected cells. Kanadaptin may not be involved in the biosynthesis and targeting of kAE1. As such, defects in kanadaptin and its interaction with kAE1 are unlikely to be involved in the pathogenesis of the inherited kidney disease, distal renal tubular acidosis (dRTA).     

Human kidney anion exchanger 1 interacts with adaptor-related protein complex 1 μ1A (AP-1 mu1A)

Kidney anion exchanger 1 (kAE1) mediates chloride (Cl⁻) and bicarbonate (HCO₃⁻) exchange at the basolateral membrane of kidney α-intercalated cells. Impaired trafficking of kAE1 leads to defect of the Cl⁻/HCO₃⁻ exchange at the basolateral membrane and failure of proton (H⁺) secretion at the apical membrane, causing a kidney disease - distal renal tubular acidosis (dRTA). To gain a better insight into kAE1 trafficking, we searched for proteins physically interacting with the C-terminal region of kAE1 (Ct-kAE1), which contains motifs crucial for intracellular trafficking, by a yeast two-hybrid (Y2H) system. An adaptor-related protein complex 1 μ1A (AP-1 mu1A) subunit was found to interact with Ct-kAE1. The interaction between either Ct-kAE1 or full-length kAE1 and AP-1 mu1A were confirmed in human embryonic kidney (HEK) 293T by co-immunoprecipitation, affinity co-purification, co-localization, yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) and GST pull-down assay. The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXØ motif. Interestingly, suppression of endogenous AP-1 mu1A in HEK 293T by small interfering RNA (siRNA) decreased membrane localization of kAE1 and increased its intracellular accumulation, suggesting for the first time that AP-1 mu1A is involved in the kAE1 trafficking of kidney α-intercalated cells.     

Yos9p and Hrd1p mediate ER retention of misfolded proteins for ER-associated degradation

The endoplasmic reticulum (ER) has an elaborate quality control system, which retains misfolded proteins and targets them to ER-associated protein degradation (ERAD). To analyze sorting between ER retention and ER exit to the secretory pathway, we constructed fusion proteins containing both folded carboxypeptidase Y (CPY) and misfolded mutant CPY (CPY*) units. Although the luminal Hsp70 chaperone BiP interacts with the fusion proteins containing CPY* with similar efficiency, a lectin-like ERAD factor Yos9p binds to them with different efficiency. Correlation between efficiency of Yos9p interactions and ERAD of these fusion proteins indicates that Yos9p but not BiP functions in the retention of misfolded proteins for ERAD. Yos9p targets a CPY*-containing ERAD substrate to Hrd1p E3 ligase, thereby causing ER retention of the misfolded protein. This ER retention is independent of the glycan degradation signal on the misfolded protein and operates even when proteasomal degradation is inhibited. These results collectively indicate that Yos9p and Hrd1p mediate ER retention of misfolded proteins in the early stage of ERAD, which constitutes a process separable from the later degradation step.     

Cln6 mutants associated with neuronal ceroid lipofuscinosis are degraded in a proteasome-dependent manner

NCLs (neuronal ceroid lipofuscinoses), a group of inherited neurodegenerative lysosomal storage diseases that predominantly affect children, are the result of autosomal recessive mutations within one of the nine cln genes. The wild-type cln gene products are composed of membrane and soluble proteins that localize to the lysosome or the ER (endoplasmic reticulum). However, the destiny of the Cln variants has not been fully characterized. To explore a possible link between ER quality control and processing of Cln mutants, we investigated the fate of two NCL-related Cln6 mutants found in patient samples (Cln6(G123D) and Cln6(M241T)) in neuronal-derived human cells. The point mutations are predicted to be in the putative transmembrane domains and most probably generate misfolded membrane proteins that are subjected to ER quality control. Consistent with this paradigm, both mutants underwent rapid proteasome-mediated degradation and complexed with components of the ER extraction apparatus, Derlin-1 and p97. In addition, knockdown of SEL1L [sel-1 suppressor of lin-12-like (Caenorhabditis elegans)], a member of an E3 ubiquitin ligase complex involved in ER protein extraction, rescued significant amounts of Cln6(G123D) and Cln6(M241T) polypeptides. The results implicate ER quality control in the instability of the Cln variants that probably contributes to the development of NCL.     

Interactions of mouse glycophorin A with the dRTA-related mutant G719D of the mouse Cl-/HCO3- exchanger Ae1

The AE1 mutation G701D, associated with recessive distal renal tubular acidosis (dRTA), produces only minimal erythroid phenotype, reflecting erythroid-specific expression of stimulatory AE1 subunit glycophorin A (GPA). GPA transgene expression could theoretically treat recessive dRTA in patients and in mice expressing cognate Ae1 mutation G719D. However, human (h) GPA and mouse (m) Gpa amino acid sequences are widely divergent, and mGpa function in vitro has not been investigated. We therefore studied in Xenopus oocytes the effects of coexpressed mGpa and hGPA on anion transport by erythroid (e) and kidney (k) isoforms of wild-type mAe1 (meAe1, mkAe1) and of mAe1 mutant G719D. Coexpression of hGPA or mGpa enhanced the function of meAe1 and mkAe1 and rescued the nonfunctional meAe1 and mkAe1 G719D mutants through increased surface expression. Progressive N-terminal truncation studies revealed a role for meAe1 amino acids 22-28 in GPA-responsiveness of meAe1 G719D. MouseN-cyto/humanTMD and humanN-cyto/mouseTMD kAE1 chimeras were active and GPA-responsive. In contrast, whereas chimera mkAe1N-cyto/hkAE1 G701DTMD was GPA-responsive, chimera hkAE1N-cyto/mkAe1 G719DTMD was GPA-insensitive. Moreover, whereas the isolated transmembrane domain (TMD) of hAE1 G701D was GPA-responsive, that of mAe1 G719D was GPA-insensitive. Thus, mGpa increases surface expression and activity of meAe1 and mkAe1. However, the G719D mutation renders certain mAe1 mutant constructs GPA-unresponsive and highlights a role for erythroid-specific meAe1 amino acids 22-28 in GPA-responsiveness.