Contrasting Functions of Calreticulin and Calnexin in Glycoprotein Folding and ER Quality Control

Molinari, Maurizio; Eriksson, Klara Kristin; Calanca, Verena; Galli, Carmela; Cresswell, Peter; Michalak, Marek; Helenius, Ari

doi:10.1016/s1097-2765(03)00494-5

Cited by 196 publications

(154 citation statements)

References 60 publications

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“…In this case, Ero1α would not be the first ER oxidative protein chaperone involved in the regulation of ER calcium homeostasis. These observations have a precedent in the functions of calreticulin (Michalak et al 2009;Molinari et al 2004), but the presence of a calcium binding domain within Ero1α itself also supports such a possibility. Further research is required to address these possibilities and also to clarify the functional relationship between the two human Ero1 protein family members.…”

Section: Discussionmentioning

confidence: 80%

“…Similarly, calreticulin inhibits uptake of calcium by inhibiting the affinity for calcium of the SERCA2b pump, but also regulates IP3-induced calcium release (Camacho and Lechleiter 1995;John et al 1998). In vivo, these functions of calreticulin may very well be more crucial for survival than its chaperone activity, since calreticulin-deficient cells have impaired Ca 2+ homoeostasis, yet only a modest decrease in protein folding (Michalak et al 2009;Molinari et al 2004). Another example of a folding enzyme regulating ER calcium content is the oxidoreductase ERp44 that interacts with cysteines of the IP3R type 1, thereby inhibiting calcium transfer to mitochondria when ER conditions are reducing (Higo et al 2005).…”

Section: Introductionmentioning

confidence: 99%

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Ero1α requires oxidizing and normoxic conditions to localize to the mitochondria-associated membrane (MAM)

Gilady

Bui

Lynes

et al. 2010

Cell Stress and Chaperones

156

115

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Protein secretion from the endoplasmic reticulum (ER) requires the enzymatic activity of chaperones and oxidoreductases that fold polypeptides and form disulfide bonds within newly synthesized proteins. The best-characterized ER redox relay depends on the transfer of oxidizing equivalents from molecular oxygen through ER oxidoreductin 1 (Ero1) and protein disulfide isomerase to nascent polypeptides. The formation of disulfide bonds is, however, not the sole function of ER oxidoreductases, which are also important regulators of ER calcium homeostasis. Given the role of human Ero1α in the regulation of the calcium release by inositol 1,4,5-trisphosphate receptors during the onset of apoptosis, we hypothesized that Ero1α may have a redox-sensitive localization to specific domains of the ER. Our results show that within the ER, Ero1α is almost exclusively found on the mitochondria-associated membrane (MAM). The localization of Ero1α on the MAM is dependent on oxidizing conditions within the ER. Chemical reduction of the ER environment, but not ER stress in general leads to release of Ero1α from the MAM. In addition, the correct localization of Ero1α to the MAM also requires normoxic conditions, but not ongoing oxidative phosphorylation.

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Section: Discussionmentioning

confidence: 80%

Section: Introductionmentioning

confidence: 99%

Ero1α requires oxidizing and normoxic conditions to localize to the mitochondria-associated membrane (MAM)

Gilady

Bui

Lynes

et al. 2010

Cell Stress and Chaperones

156

115

View full text Add to dashboard Cite

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“…First, the co-translational interaction with chaperones generates a folding intermediate that is committed to folding. For example, the HA folding process is strongly dependent on calnexin interaction (18,19). HA interacts co-translationally with calnexin, and the lack of its TMD does not drastically affect the post-translational calnexin interaction (20).…”

Section: Discussionmentioning

confidence: 99%

Productive Folding of Tyrosinase Ectodomain Is Controlled by the Transmembrane Anchor

Popescu

Mares

Zdrentu

et al. 2006

Journal of Biological Chemistry

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Transmembrane domains (TMDs) are known as structural elements required for the insertion into the membrane of integral membrane proteins. We have provided here an example showing that the presence of the TMD is compulsory for the productive folding pathway of a membrane-anchored glycoprotein. Tyrosinase, a type I transmembrane protein whose insertion into the melanosomal membrane initiates melanin synthesis, is misfolded and degraded when expressed as a truncated polypeptide. We used constructs of tyrosinase ectodomain fused with chimeric TMDs or glycosylphosphatidylinositol anchor to gain insights into how the TMD enables the productive folding pathway of the ectodomain. We found that in contrast to the soluble constructs, the membrane-anchored chimeras fold into the native conformation, which allows their endoplasmic reticulum exit. They recruit calnexin to monitor their productive folding pathway characterized by the posttranslational formation of buried disulfides. Lacking calnexin assistance, the truncated mutant is arrested in an unstable conformation bearing exposed disulfides. We showed that the transmembrane anchor of a protein may crucially, albeit indirectly, control the folding pathway of the ectodomain.

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“…3a) in the pulse medium. Because HA needs CNX for folding 11 , a relatively large fraction of HA aggregated in the control experiment without DTT (Fig. 4a).…”

Section: Reduced Ha Cannot Fold In a Cell Lysatementioning

confidence: 99%

“…It folds in the ER, trimerizes and is transported to the plasma membrane, where it is incorporated into new virions. The folding of HA has been studied extensively both in complete cells [5][6][7][8][9][10][11] and in microsomes 12,13 , using radioactive labeling of newly synthesized HA or truncated ribosome-bound nascent chains 14 . Although these studies yielded fairly detailed information on the HA folding process, further molecular insight would require biophysical studies on purified protein, which would disregard the role of the cell.…”

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confidence: 99%

A critical step in the folding of influenza virus HA determined with a novel folding assay

Maggioni

Liscaljet

Braakman

2005

Nat Struct Mol Biol

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Most principles of protein folding emerged from refolding studies in vitro on small, soluble proteins, because large ones tend to misfold and aggregate. We developed a folding assay allowing the study of large proteins in detergent such that the extent of cellular assistance required for proper folding can be determined. We identified a critical step in the in vivo folding pathway of influenza virus hemagglutinin (HA). Only the formation of the first few disulfides in the top domain of HA required the intact endoplasmic reticulum. After that, HA proceeded to fold efficiently in a very dilute solution, despite its size and complexity. This study paves the way for detailed structural analyses during the folding of complex proteins.Protein folding is the process by which linear polypeptide chains acquire their biologically active three-dimensional conformation. Although all information needed to reach the native conformation is encoded by the amino acid sequence 1 , protein folding inside the cell generally is assisted by molecular chaperones and folding enzymes 2-4 . They mainly protect nascent chains and newly synthesized proteins from misfolding and aggregation. Chaperones and folding enzymes reside in almost every compartment of eukaryotic cells.The endoplasmic reticulum (ER) is highly specialized for the folding of membrane and secretory proteins. One of the bestcharacterized model proteins used to study protein folding in the ER is the influenza virus HA. This protein is not only crucial for influenza virus infection but also an important target for the immune system. HA is a multidomain glycoprotein of 84 kDa with six disulfide bonds and seven N-linked glycans. It folds in the ER, trimerizes and is transported to the plasma membrane, where it is incorporated into new virions. The folding of HA has been studied extensively both in complete cells [5][6][7][8][9][10][11] and in microsomes 12,13 , using radioactive labeling of newly synthesized HA or truncated ribosome-bound nascent chains 14 . Although these studies yielded fairly detailed information on the HA folding process, further molecular insight would require biophysical studies on purified protein, which would disregard the role of the cell.To start bridging the gap between folding studies in vitro and in intact cells, we developed an assay that allows the study of post-translational HA folding in a cell-free system without membranes. We replaced the chase period in the well-established pulse-chase folding assay 6 with a folding analysis in solution: the protein is translated and translocated in intact cells, after which post-translational folding is studied in a detergent cell lysate. Using this 'in vitro chase' assay, we found that HA can fold in a detergent cell lysate without ER assistance, provided that the first folding step occurs in an intact ER. RESULTS HA can fold in a detergent cell lysateWhen studied with the pulse-chase assay in intact cells, newly synthesized HA ran as a single band in a reducing SDS-PAGE gel (Fig. 1a, R) and as three ban...

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Contrasting Functions of Calreticulin and Calnexin in Glycoprotein Folding and ER Quality Control

Cited by 196 publications

References 60 publications

Ero1α requires oxidizing and normoxic conditions to localize to the mitochondria-associated membrane (MAM)

Ero1α requires oxidizing and normoxic conditions to localize to the mitochondria-associated membrane (MAM)

Productive Folding of Tyrosinase Ectodomain Is Controlled by the Transmembrane Anchor

A critical step in the folding of influenza virus HA determined with a novel folding assay

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