Golgi Apparatus Immunolocalization of Endomannosidase Suggests Post-Endoplasmic Reticulum Glucose Trimming: Implications for Quality Control

Zuber, Christian; Spiro, Mary Jane; Guhl, Bruno; Spiro, Robert G.; Roth, Jürgen

doi:10.1091/mbc.11.12.4227

Cited by 103 publications

(108 citation statements)

References 76 publications

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“…Consistent with this, expression of poliovirus 2B was reported to accumulate secretory glycoproteins in an endoglycosidase H-sensitive glycosylation state (28). Accumulation of improperly glycosylated proteins in the Golgi complex is in agreement with the role of Golgi complex in the quality control of protein folding (29,30). The inhibition of transport by 2B may be due to its effect on Golgi pH, similar as has been described for the Na ϩ /H ϩ -ionophore monensin (20).…”

Section: Discussionsupporting

confidence: 71%

The Coxsackievirus 2B Protein Increases Efflux of Ions from the Endoplasmic Reticulum and Golgi, thereby Inhibiting Protein Trafficking through the Golgi

Jong¹,

Visch

Mattia³

et al. 2006

Journal of Biological Chemistry

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Coxsackievirus infection leads to a rapid reduction of the filling state of the endoplasmic reticulum (ER) and Golgi Ca 2؉ stores. The coxsackievirus 2B protein, a small membrane protein that localizes to the Golgi and to a lesser extent to the ER, has been proposed to play an important role in this effect by forming membrane-integral pores, thereby increasing the efflux of Ca 2؉ from the stores. Here, evidence is presented that supports this idea and that excludes the possibility that 2B reduces the uptake of Ca 2؉ into the stores. Measurement of intra-organelle-free Ca 2؉ in permeabilized cells revealed that the ability of 2B to reduce the Ca 2؉ filling state of the stores was preserved at steady ATP. Biochemical analysis in a cellfree system further showed that 2B had no adverse effect on the activity of the sarco/endoplasmic reticulum calcium ATPase, the Ca 2؉ -ATPase that transports Ca 2؉ from the cytosol into the stores. To investigate whether 2B specifically affects Ca 2؉ homeostasis or other ion gradients, we measured the lumenal Golgi pH. Expression of 2B resulted in an increased Golgi pH, indicative for the efflux of H ؉ from the Golgi lumen. Together, these data support a model that 2B increases the efflux of ions from the ER and Golgi by forming membrane-integral pores. We have demonstrated that a major consequence of this activity is the inhibition of protein trafficking through the Golgi complex.Enteroviruses (e.g. poliovirus, coxsackievirus, ECHOvirus) belong to the family of Picornaviridea, a large family of nonenveloped, cytolytic viruses that contain a single-stranded RNA genome of positive polarity. Upon infection, enteroviruses induce a number of dramatic alterations in their host cell, which serve to create the appropriate conditions for viral RNA replication and/or prevent antiviral host cell responses. One of these alterations is the modification of intracellular Ca 2ϩ homeostasis. We have previously shown that infection of HeLa cells with coxsackievirus results in a reduction of the amount of Ca 2ϩ that can be released from the intracellular stores using thapsigargin, an inhibitor of the sarco/endoplasmic reticulum calcium ATPase (SERCA), 2 the Ca 2ϩ -ATPase that transports Ca 2ϩ from the cytosol into the stores. In addition, a gradual increase in the cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] cyt ) was observed due to the influx of extracellular Ca 2ϩ (1). The enterovirus 2B protein, one of the nonstructural proteins involved in viral RNA replication, plays a major role in the alterations in intracellular Ca 2ϩ homeostasis that take place in enterovirus-infected cells (1, 2). The mechanism by which 2B, or its precursor 2BC, exerts its effects is largely unknown. Ca 2ϩ homeostasis in the intracellular stores (i.e. endoplasmic reticulum (ER) and Golgi) is the net result of the activity of the SERCA on the one hand and the continuous passive Ca 2ϩ leak from these organelles that exists under normal conditions on the other hand (3). Thus, the reductions in the Ca 2ϩ filling state of the stores ...

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Section: Discussionsupporting

confidence: 71%

The Coxsackievirus 2B Protein Increases Efflux of Ions from the Endoplasmic Reticulum and Golgi, thereby Inhibiting Protein Trafficking through the Golgi

Jong¹,

Visch

Mattia³

et al. 2006

Journal of Biological Chemistry

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“…Endo-α-mannosidase is a resident of the cis/medial compartment of the Golgi apparatus and pre-Golgi intermediates, and is a membrane-associated protein that is difficult to recombinantly express in soluble form (29). The bacterial orthologs from B. thetaiotaomicron and B. xylanisolvens are soluble proteins and may have been acquired by horizontal gene transfer because these organisms are common and beneficial components of the human gut (30).…”

Section: Discussionmentioning

confidence: 99%

Structural and mechanistic insight into N-glycan processing by endo-α-mannosidase

Thompson

Williams

Hakki

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

120

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N-linked glycans play key roles in protein folding, stability, and function. Biosynthetic modification of N-linked glycans, within the endoplasmic reticulum, features sequential trimming and readornment steps. One unusual enzyme, endo-α-mannosidase, cleaves mannoside linkages internally within an N-linked glycan chain, short circuiting the classical N-glycan biosynthetic pathway. Here, using two bacterial orthologs, we present the first structural and mechanistic dissection of endo-α-mannosidase. Structures solved at resolutions 1.7–2.1 Å reveal a ( β / α ) 8 barrel fold in which the catalytic center is present in a long substrate-binding groove, consistent with cleavage within the N-glycan chain. Enzymatic cleavage of authentic Glc 1/3 Man 9 GlcNAc 2 yields Glc 1/3 -Man. Using the bespoke substrate α-Glc-1,3-α-Man fluoride, the enzyme was shown to act with retention of anomeric configuration. Complexes with the established endo-α-mannosidase inhibitor α-Glc-1,3-deoxymannonojirimycin and a newly developed inhibitor, α-Glc-1,3-isofagomine, and with the reducing-end product α-1,2-mannobiose structurally define the -2 to +2 subsites of the enzyme. These structural and mechanistic data provide a foundation upon which to develop new enzyme inhibitors targeting the hijacking of N-glycan synthesis in viral disease and cancer.

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“…In the end, both pathways meet in the ER where ERAD takes place (40,41). In mammalian cells, the quality control machinery proteins glucosidase II, glucosyltransferase and calreticulin have been shown to be present beyond the ER in pre-Golgi intermediates (42)(43)(44), indicating their function as post-ER quality control checkpoints.…”

Section: Discussionmentioning

confidence: 99%

EDEM1 reveals a quality control vesicular transport pathway out of the endoplasmic reticulum not involving the COPII exit sites

Zuber

Cormier

Guhl

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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104

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Immature and nonnative proteins are retained in the endoplasmic reticulum (ER) by the quality control machinery. Folding-incompetent glycoproteins are eventually targeted for ER-associated protein degradation (ERAD). EDEM1 (ER degradation-enhancing ␣-mannosidaselike protein 1), a putative mannose-binding protein, targets misfolded glycoproteins for ERAD. We report that endogenous EDEM1 exists mainly as a soluble glycoprotein. By high-resolution immunolabeling and serial section analysis, we find that endogenous EDEM1 is sequestered in buds that form along cisternae of the rough ER at regions outside of the transitional ER. They give rise to Ϸ150-nm vesicles scattered throughout the cytoplasm that are lacking a recognizable COPII coat. About 87% of the immunogold labeling was over the vesicles and Ϸ11% over the ER lumen. Some of the EDEM1 vesicles also contain Derlin-2 and the misfolded Hong Kong variant of ␣-1-antitrypsin, a substrate for EDEM1 and ERAD. Our results demonstrate the existence of a vesicle budding transport pathway out of the rough ER that does not involve the canonical transitional ER exit sites and therefore represents a previously unrecognized passageway to remove potentially harmful misfolded luminal glycoproteins from the ER.electron microscopy ͉ protein misfolding ͉ protein quality control T he folding state of newly synthesized glycoproteins in the endoplasmic reticulum (ER) is monitored by a quality control machinery (1). Increased formation of misfolded proteins disturbs ER homeostasis resulting in protein degradation, as well as cell damage and death. This is the cause of many human diseases including cystic fibrosis, ␣-1-antitrypsin deficiency, renal diabetes insipidus, and congenital goiter (2, 3).Orderly occurring processes can be distinguished during the life and death of a folding-incompetent glycoprotein. The first involves recognition by the quality control machinery (4-6). The lectin chaperones calnexin/calreticulin retain nonnative conformers with monoglucosylated glycans (5, 7). Mannose-trimmed glycans generated by ER-mannosidases appear to represent a quality control tag for routing misfolded glycoproteins to the ER-associated degradation (ERAD) (8-12). The current view is that misfolded, mannosetrimmed glycoproteins are then retrotranslocated to the cytoplasm, where they are ubiquitinated and deglycosylated before proteasomal degradation (13).EDEM1 (ER degradation-enhancing ␣-mannosidase-like protein 1) and its yeast ortholog Htm1p/Mnl1p are putative mannosebinding proteins (14-18) that are transcriptionally induced by ER stress (14,17,18). They are believed to target misfolded glycoproteins for proteasomal degradation by removing them from the calnexin/calreticulin cycle (14-17). The overexpression of EDEM1 results in the accelerated proteasomal degradation of ERAD substrates such as the Hong Kong variant of ␣-1-antitrypsin (HK A1AT) and a luminal variant of beta-secretase, whereas the deletion or knockdown of EDEM1/Htm1p reduces the degradation rates of the ERAD substrates ...

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Golgi Apparatus Immunolocalization of Endomannosidase Suggests Post-Endoplasmic Reticulum Glucose Trimming: Implications for Quality Control

Cited by 103 publications

References 76 publications

The Coxsackievirus 2B Protein Increases Efflux of Ions from the Endoplasmic Reticulum and Golgi, thereby Inhibiting Protein Trafficking through the Golgi

The Coxsackievirus 2B Protein Increases Efflux of Ions from the Endoplasmic Reticulum and Golgi, thereby Inhibiting Protein Trafficking through the Golgi

Structural and mechanistic insight into N-glycan processing by endo-α-mannosidase

EDEM1 reveals a quality control vesicular transport pathway out of the endoplasmic reticulum not involving the COPII exit sites

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