Dataset Information

ER entry pathway and glycosylation of GPI-anchored proteins are determined by N-terminal signal sequence and C-terminal GPI-attachment sequence.

ABSTRACT: Newly synthesized proteins in the secretory pathway, including glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs), need to be correctly targeted and imported into the endoplasmic reticulum (ER) lumen. GPI-APs are synthesized in the cytosol as preproproteins, which contain an N-terminal signal sequence (SS), mature protein part, and C-terminal GPI-attachment sequence (GPI-AS), and translocated into the ER lumen where SS and GPI-AS are removed, generating mature GPI-APs. However, how various GPI-APs are translocated into the ER lumen in mammalian cells is unclear. Here, we investigated the ER entry pathways of GPI-APs using a panel of KO cells defective in each signal recognition particle-independent ER entry pathway-namely, Sec62, GET, or SND pathway. We found GPI-AP CD59 largely depends on the SND pathway for ER entry, whereas prion protein (Prion) and LY6K depend on both Sec62 and GET pathways. Using chimeric Prion and LY6K constructs in which the N-terminal SS or C-terminal GPI-AS was replaced with that of CD59, we revealed that the hydrophobicity of the SSs and GPI-ASs contributes to the dependence on Sec62 and GET pathways, respectively. Moreover, the ER entry route of chimeric Prion constructs with the C-terminal GPI-ASs replaced with that of CD59 was changed to the SND pathway. Simultaneously, their GPI structures and which oligosaccharyltransferase isoforms modify the constructs were altered without any amino acid change in the mature protein part. Taking these findings together, this study revealed N- and C-terminal sequences of GPI-APs determine the selective ER entry route, which in turn regulates subsequent maturation processes of GPI-APs.

SUBMITTER: Hirata T

PROVIDER: S-EPMC9520029 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

ER entry pathway and glycosylation of GPI-anchored proteins are determined by N-terminal signal sequence and C-terminal GPI-attachment sequence.

Hirata Tetsuya T Yang Jing J Tomida Seita S Tokoro Yuko Y Kinoshita Taroh T Fujita Morihisa M Kizuka Yasuhiko Y

The Journal of biological chemistry 20220831 10

Newly synthesized proteins in the secretory pathway, including glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs), need to be correctly targeted and imported into the endoplasmic reticulum (ER) lumen. GPI-APs are synthesized in the cytosol as preproproteins, which contain an N-terminal signal sequence (SS), mature protein part, and C-terminal GPI-attachment sequence (GPI-AS), and translocated into the ER lumen where SS and GPI-AS are removed, generating mature GPI-APs. However, how v ...[more]

PMID: 36055406

Similar Datasets

Project description:BackgroundS. cerevisiae Yps1 is the prototypical aspartic endopeptidase of the fungal yapsin family. This glycosylphosphatidylinositol (GPI) anchored enzyme was recently shown to be involved in the shedding of the GPI proteins Utr2, Gas1 and itself. It was also proposed to be part of a novel quality control mechanism that eliminates excess and/or misfolded GPI proteins. What regulates its shedding activity at the cell surface is however poorly understood. Yps1 is initially synthesized as a zymogen requiring proteolytic activation to remove a pro-peptide and further processing within a large insertion loop (N-entrance loop) generates a two-subunit endopeptidase. To investigate the role of this loop on its shedding activity, which typically takes place within Ser/Thr-rich domains, it was replaced with the short peptide found at the analogous position in Yps3. We also tested whether O-glycosylation might protect against proteolytic processing by Yps1.ResultsWe show here that replacement of the N-entrance loop (N-ent loop) of Yps1 generates a single chain endopeptidase that undergoes partial (pH 6.0) or complete (pH 3.0) pro-peptide removal. At both pH, the shedding activity of the chimeric endopeptidase (Yps1-DL) toward Gas1 and itself is strongly and drastically increased, respectively. A direct correlation between endoproteolytic cleavage of this loop in native Yps1 and its shedding is observed. The Yps1-dependent shedding of two model GPI proteins (Gas1 and Yps1) is also stimulated by the absence of the O-mannosyltransferases, Pmt4 and Pmt2 respectively, involved in O-glycosylation of their Ser/Thr-rich domains. Under these conditions, some Yps1-independent shedding is also observed.ConclusionsPartial pro-peptide removal is essential to produce a functional Yps1 endopeptidase. The Yps1 N-ent loop plays a major role in regulating the shedding activity of the endopeptidase, most likely by limiting access to the active site, and its cleavage in native Yps1 is associated with its shedding. O-glycosylation protects against Yps1-dependent and -independent shedding of GPI proteins. It is postulated that hypoglycosylation of cell surface proteins, which may occur for misfolded proteins that escaped the ER-associated degradation, might target their elimination through shedding by Yps1 and possibly other yapsin members.

Dataset Information

ER entry pathway and glycosylation of GPI-anchored proteins are determined by N-terminal signal sequence and C-terminal GPI-attachment sequence.

Publications

ER entry pathway and glycosylation of GPI-anchored proteins are determined by N-terminal signal sequence and C-terminal GPI-attachment sequence.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets