Human endoplasmic reticulum mannosidase I is subject to regulated proteolysis

Ying Wu, Daniel J. Termine, Matthew T. Swulius, Kelley W. Moremen, Richard N. Sifers

Research output: Contribution to journalArticle

44 Citations (Scopus)

Abstract

In the early secretory pathway, opportunistic cleavage of asparagine-linked oligosaccharides by endoplasmic reticulum (ER) mannosidase I targets misfolded glycoproteins for dislocation into the cytosol and destruction by 26 S proteasomes. The low basal concentration of the glycosidase is believed to coordinate the glycan cleavage with prolonged conformation-based ER retention, ensuring that terminally misfolded glycoproteins are preferentially targeted for destruction. Herein the intracellular fate of human ER mannosidase I was monitored to determine whether a post-translational process might contribute to the regulation of its intracellular concentration. The transiently expressed recombinant human glycosidase was subject to rapid intracellular turnover in mouse hepatoma cells, as was the endogenous mouse ortholog. Incubation with either chloroquine or leupeptin, but not lactacystin, led to intracellular stabilization, implicating the involvement of lysosomal acid hydrolases. Inhibition of protein synthesis with cycloheximide led to intracellular depletion of the glycosidase and concomitant ablation of asparagine-linked glycoprotein degradation, confirming the physiologic relevance of the destabilization process. Metabolic incorporation of radiolabeled phosphate, detection by anti-phosphoserine antiserum, and the stabilizing effect of general serine kinase inhibition implied that ER mannosidase I is subjected to regulated proteolysis. Stabilization in response to genetically engineered removal of the amino-terminal cytoplasmic tail, a postulated regulatory domain, and colocalization of green fluorescent protein fusion proteins with Lamp1 provided two additional lines of evidence to support the hypothesis. A model is proposed in which proteolytically driven checkpoint control of ER mannosidase I contributes to the establishment of an equitable glycoprotein quality control standard by which the efficiency of asparagine-linked glycoprotein conformational maturation is measured.

Original languageEnglish (US)
Pages (from-to)4841-4849
Number of pages9
JournalJournal of Biological Chemistry
Volume282
Issue number7
DOIs
StatePublished - Feb 16 2007

Fingerprint

mannosyl-oligosaccharide 1,2-alpha-mannosidase
Proteolysis
Endoplasmic Reticulum
Glycoproteins
Glycoside Hydrolases
Asparagine
Stabilization
Phosphoserine
Secretory Pathway
Protein-Serine-Threonine Kinases
Chloroquine
Hydrolases
Proteasome Endopeptidase Complex
Cycloheximide
Ablation
Green Fluorescent Proteins
Oligosaccharides
Quality Control
Cytosol
Quality control

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Wu, Ying ; Termine, Daniel J. ; Swulius, Matthew T. ; Moremen, Kelley W. ; Sifers, Richard N. / Human endoplasmic reticulum mannosidase I is subject to regulated proteolysis. In: Journal of Biological Chemistry. 2007 ; Vol. 282, No. 7. pp. 4841-4849.
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Human endoplasmic reticulum mannosidase I is subject to regulated proteolysis. / Wu, Ying; Termine, Daniel J.; Swulius, Matthew T.; Moremen, Kelley W.; Sifers, Richard N.

In: Journal of Biological Chemistry, Vol. 282, No. 7, 16.02.2007, p. 4841-4849.

Research output: Contribution to journalArticle

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AU - Wu, Ying

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AU - Swulius, Matthew T.

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