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PMID: 15358861 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

A complex between peptide:N-glycanase and two proteasome-linked proteins suggests a mechanism for the degradation of misfolded glycoproteins.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 101 ·No. 38 ·2004-09-21 ·Pages 13774-9

Katiyar S, Li G, Lennarz WJ

Abstract

Peptide:N-glycanase (PNGase) has been proposed to participate in the proteasome-dependent glycoprotein degradation pathway. The finding that yeast PNGase interacts with the 19S proteasome subunit through the protein Rad23 supports this hypothesis. In this report, we have used immunofluorescence, subcellular fractionation, coimmunoprecipitation, and in vitro GST pull-down techniques for detecting intracellular localization and interactions of PNGase, HR23B, and S4 by using human (h) and mouse (m) homologs. Immunofluorescence studies revealed that hPNGase, hHR23B, and hS4 are present in close proximity to the endoplasmic reticulum (ER) when calnexin was used as an ER marker in HeLa cells. Subcellular fractionation suggests not only cytoplasmic but also ER association of hPNGase in HeLa cells. Immunoprecipitation analysis revealed the interaction of h/mPNGase with the 19S proteasome subunit, hS4, through hHR23B. Using an in vitro GST pull-down assay, we also have shown that recombinant mPNGase requires its N terminus and middle domain for interaction with mHR23B. Finally, using misfolded yeast carboxypeptidase Y and chicken ovalbumin as glycoprotein substrates, we have established that mHR23B acts as a receptor for deglycosylated proteins. Based on this finding, we propose that after deglycosylation of misfolded glycoproteins by PNGase, the aglyco forms of these proteins are recognized by HR23B and targeted for degradation.

MeSH Terms
Glycoproteins/metabolism Glycosylation HeLa Cells Humans Kinetics Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/metabolism Proteasome Endopeptidase Complex Protein Conformation Protein Folding Recombinant Fusion Proteins/metabolism Recombinant Proteins/metabolism Sequence Deletion Substrate Specificity Transfection
Chemicals
Glycoproteins Recombinant Fusion Proteins Recombinant Proteins Proteasome Endopeptidase Complex Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Katiyar Samiksha
Department of Biochemistry and Cell Biology and Institute for Cell and Developmental Biology, Stony Brook University, Stony Brook, NY 11794-5215, USA.
Li Guangtao
Lennarz William J
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.0405663101
Published
2004-09-21
Epub
2004-00-09
Pages
13774-9
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC518832
Subset
IM
Grants
NIGMS NIH HHS · GM33814 · United States
External Links
PubMed source DOI: 10.1073/pnas.0405663101 PMC Full Text View journal details
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