ABSTRACT
The rough ER (rER) plays a central role in the biogenesis of most extracellular and many organellar proteins in eukaryotic cells. Cells that are specialized in protein secretion, such as pancreatic cells, are particularly rich in rER. In the process of cell homogenization, the rER is converted into ribosome-studded vesicles, the so-called rough microsomes. Here we report on a membrane proteomic analysis of canine pancreatic rough microsomes. Special emphasis was placed on components involved in the various aspects of protein biogenesis, such as protein transport, protein folding, protein modification, and protein degradation. Our results indicate that the Hsp70-chaperone network that is present in the pancreatic ER is even more complex than previously thought, and suggest that the pancreatic rER has a significant capacity for protein degradation.
Subject(s)
Endoplasmic Reticulum, Rough/chemistry , HSP40 Heat-Shock Proteins/analysis , Microsomes/chemistry , Pancreas/chemistry , Proteome/analysis , Amino Acid Sequence , Animals , Cell Line, Tumor , Cell-Free System , Dogs , Endoplasmic Reticulum, Rough/metabolism , HSP40 Heat-Shock Proteins/chemistry , HSP40 Heat-Shock Proteins/metabolism , Humans , Liver/chemistry , Liver/metabolism , Mass Spectrometry , Microsomes/metabolism , Molecular Sequence Data , Pancreas/metabolism , Proteome/metabolism , Sequence AlignmentABSTRACT
Previous studies have shown that the rough endoplasmic reticulum (ER) contains nascent precursor polypeptide gated channels. Circumstantial evidence suggests that these channels are formed by the Sec61p complex. We reconstituted the purified Sec61p complex in a lipid bilayer and characterized its dynamics and regulation. The Sec61p complex is sufficient to form the precursor polypeptide activated channel under co- and posttranslational transport conditions. Activity of the Sec61p channel in both transport modes is induced by direct interaction with precursor protein. The Sec61p complex comprises a highly dynamic pore covering conductances corresponding to channel openings from approximately 6 to 60 A. Its properties are indistinguishable from those we observed with native ER channels, directly demonstrating that these channels are formed by the Sec61p complex.
Subject(s)
Endoplasmic Reticulum, Rough/metabolism , Eukaryotic Cells/metabolism , Heat-Shock Proteins , Intracellular Membranes/metabolism , Ion Channels/metabolism , Membrane Proteins/metabolism , Protein Biosynthesis , Protein Transport/physiology , ADP Ribose Transferases/pharmacology , Animals , Bacterial Toxins/pharmacology , Carrier Proteins/metabolism , Carrier Proteins/pharmacology , Endoplasmic Reticulum Chaperone BiP , Exotoxins/pharmacology , Humans , Ion Channels/drug effects , Macromolecular Substances , Membrane Proteins/drug effects , Molecular Chaperones/metabolism , Molecular Chaperones/pharmacology , Molecular Structure , Peptides/metabolism , Protein Processing, Post-Translational/physiology , Protein Transport/drug effects , SEC Translocation Channels , Virulence Factors/pharmacology , Pseudomonas aeruginosa Exotoxin AABSTRACT
Recently, the homolog of yeast protein Sec63p was identified in dog pancreas microsomes. This pancreatic DnaJ-like protein was shown to be an abundant protein, interacting with both the Sec61p complex and lumenal DnaK-like proteins, such as BiP. The pancreatic endoplasmic reticulum contains a second DnaJ-like membrane protein, which had been termed Mtj1p in mouse. Mtj1p is present in pancreatic microsomes at a lower concentration than Sec63p but has a higher affinity for BiP. In addition to a lumenal J-domain, Mtj1p contains a single transmembrane domain and a cytosolic domain which is in close contact with translating ribosomes and appears to have the ability to modulate translation. The interaction with ribosomes involves a highly charged region within the cytosolic domain of Mtj1p. We propose that Mtj1p represents a novel type of co-chaperone, mediating transmembrane recruitment of DnaK-like chaperones to ribosomes and, possibly, transmembrane signaling between ribosomes and DnaK-like chaperones of the endoplasmic reticulum.
