Intracellular degradation of unassembled asialoglycoprotein receptor subunits: a pre-Golgi, nonlysosomal endoproteolytic cleavage.
The human asialoglycoprotein receptor is a heterooligomer of the two homologous subunits H1 and H2. As occurs for other oligomeric receptors, not all of the newly made subunits are assembled in the RER into oligomers and some of each chain is degraded. We studied the degradation of the unassembled H2 subunit in fibroblasts that only express H2 (45,000 mol wt) and degrade all of it. After a 30 min lag, H2 is degraded with a half-life of 30 min. We identified a 35-kD intermediate in H2 degradation; it is the COOH-terminal, exoplasmic domain of H2. After a 90-min chase, all remaining intact H2 and the 35-kD fragment were endoglycosidase H sensitive, suggesting that the cleavage generating the 35-kD intermediate occurs without translocation to the medial Golgi compartment. Treatment of cells with leupeptin, chloroquine, or NH4Cl did not affect H2 degradation. Monensin slowed but did not block degradation. Incubation at 18-20 degrees C slowed the degradation dramatically and caused an increase in intracellular H2, suggesting that a membrane trafficking event occurs before H2 is degraded. Immunofluorescence microscopy of cells with or without an 18 degrees C preincubation showed a colocalization of H2 with the ER and not with the Golgi complex. We conclude that H2 is not degraded in lysosomes and never reaches the medial Golgi compartment in an intact form, but rather degradation is initiated in a pre-Golgi compartment, possibly part of the ER. The 35-kD fragment of H2 may define an initial proteolytic cleavage in the ER.
Amara
J F
JF
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142.
Lederkremer
G
G
Lodish
H F
HF
D000118:Acetylglucosaminidase / Q000494:pharmacology; D000818:Animals; D037263:Asialoglycoprotein Receptor; D002460:Cell Line; D002738:Chloroquine / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000378:metabolism*; D006207:Half-Life; D007976:Leupeptins / Q000494:pharmacology; D008247:Lysosomes / Q000378:metabolism; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D008985:Monensin / Q000494:pharmacology; D011971:Receptors, Immunologic / Q000378:metabolism*; D013696:Temperature
D037263:Asialoglycoprotein Receptor; D007976:Leupeptins; D011971:Receptors, Immunologic; D002738:Chloroquine; D008985:Monensin; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; C032854:leupeptin