ABSTRACT
Posttranslational protein translocation across the membrane of the endoplasmic reticulum is mediated by the Sec complex. This complex includes a transmembrane channel formed by multiple copies of the Sec61 protein. Translocation of a polypeptide begins when the signal sequence binds at a specific site within the channel. Binding results in the insertion of the substrate into the channel, possibly as a loop with a small segment exposed to the lumen. While bound, the signal sequence is in contact with both protein components of the channel and the lipid of the membrane. Subsequent movement of the polypeptide through the channel occurs when BiP molecules interact transiently with a luminal domain of the Sec complex, hydrolyze ATP, and bind to the substrate. Bound BiP promotes translocation by preventing the substrate from diffusing backwards through the channel, and thus acts as a molecular ratchet.
Subject(s)
Endoplasmic Reticulum/metabolism , Protein Processing, Post-Translational , Animals , Humans , Intracellular Membranes/metabolism , Mitochondria/metabolism , Protein Sorting Signals/metabolismABSTRACT
Proteins of the Hsp70 family of ATPases interact with a conserved domain of their J-protein partners, the J-domain, to function in numerous cellular processes. We have studied the interaction of BiP, an Hsp70 family member in the lumen of the endoplasmic reticulum, with the J-domain of Sec63p, a component of the Sec complex involved in post-translational protein translocation across the endoplasmic reticulum membrane. In a real-time solid phase binding assay, BiP binds to the immobilized Sec complex or to a fusion protein of the J-domain and glutathione S-transferase in a reaction that requires ATP hydrolysis. In the final complex, BiP is bound in the ADP form with its peptide binding pocket occupied. An intact peptide binding pocket is required for this interaction. Our experiments suggest that the activation of BiP by the J-domain involves a transient contact between these components, and that in the absence of physiological substrates, J-activated BiP binds even to the J-proteins themselves.
Subject(s)
Carrier Proteins/metabolism , Endoplasmic Reticulum/metabolism , Fungal Proteins/metabolism , Heat-Shock Proteins , Membrane Proteins/metabolism , Membrane Transport Proteins , Molecular Chaperones/metabolism , Saccharomyces cerevisiae Proteins , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Base Sequence , Carrier Proteins/genetics , DNA Primers , Endoplasmic Reticulum Chaperone BiP , Enzyme Activation , Fungal Proteins/chemistry , Glutathione Transferase/metabolism , Hydrolysis , Membrane Proteins/chemistry , Molecular Chaperones/genetics , Mutagenesis , Peptides/metabolism , Protein BindingABSTRACT
We have addressed the mechanism by which proteins are posttranslationally transported across the membrane of the yeast endoplasmic reticulum (ER). We demonstrate that BiP (Kar2p), a member of the Hsp70 family resident in the ER lumen, acts as a molecular ratchet during translocation of the secretory protein prepro-alpha factor through the channel formed by the Sec complex. Multiple BiP molecules associate with each translocation substrate following interaction with the J domain of the Sec63p component of the Sec complex. Bound BiP minimizes passive backward movements of the substrate through the channel, and BiP's subsequent dissociation results in a free polypeptide in the ER lumen. Antibodies against the substrate can replace BiP, indicating that a Brownian ratchet is sufficient to achieve translocation.
Subject(s)
Endoplasmic Reticulum/metabolism , Fungal Proteins/metabolism , HSP70 Heat-Shock Proteins/metabolism , Protein Precursors/metabolism , Protein Processing, Post-Translational , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Cloning, Molecular , Escherichia coli , Fungal Proteins/chemistry , HSP70 Heat-Shock Proteins/chemistry , Intracellular Membranes/metabolism , Kinetics , Mating Factor , Molecular Sequence Data , Peptides/genetics , Protein Binding , Protein Biosynthesis , Recombinant Proteins/metabolismSubject(s)
Membrane Proteins/chemistry , Proteins/metabolism , Biological Transport , Cell Membrane/chemistry , Cell Membrane/metabolism , Endoplasmic Reticulum/chemistry , Endoplasmic Reticulum/metabolism , Membrane Proteins/metabolism , Models, Biological , Protein Biosynthesis/physiology , Protein Sorting Signals/physiology , SEC Translocation ChannelsABSTRACT
Unlike properly folded and assembled proteins, most misfolded and incompletely assembled proteins are retained in the endoplasmic reticulum of mammalian cells and degraded without transport to the Golgi complex. To analyze the mechanisms underlying this unique sorting process and its fidelity, the fate of C-terminally truncated fragments of influenza hemagglutinin was determined. An assortment of different fragments was generated by adding puromycin at low concentrations to influenza virus-infected tissue culture cells. Of the fragments generated, < 2% was secreted, indicating that the system for detecting defects in newly synthesized proteins is quite stringent. The majority of secreted species corresponded to folding domains within the viral spike glycoprotein. The retained fragments acquired a partially folded structure with intra-chain disulfide bonds and conformation-dependent antigenic epitopes. They associated with two lectin-like endoplasmic reticulum chaperones (calnexin and calreticulin) but not BiP/GRP78. Inhibition of the association with calnexin and calreticulin by the addition of castanospermine significantly increased fragment secretion. However, it also caused association with BiP/GRP78. These results indicated that the association with calnexin and calreticulin was involved in retaining the fragments. They also suggested that BiP/GRP78 could serve as a backup for calnexin and calreticulin in retaining the fragments. In summary, the results showed that the quality control system in the secretory pathway was efficient and sensitive to folding defects, and that it involved multiple interactions with endoplasmic reticulum chaperones.
Subject(s)
Proteins/metabolism , Animals , Antigens/immunology , Biological Transport, Active/physiology , CHO Cells , Calcium-Binding Proteins/physiology , Calnexin , Calreticulin , Cricetinae , Disulfides , Enzyme Inhibitors/pharmacology , Heat-Shock Proteins/physiology , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Hemagglutinin Glycoproteins, Influenza Virus/drug effects , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Indolizines/pharmacology , Peptide Fragments/chemistry , Peptide Fragments/immunology , Peptide Fragments/metabolism , Protein Synthesis Inhibitors/pharmacology , Puromycin/pharmacology , Ribonucleoproteins/physiology , Sequence Deletion/drug effects , Sequence Deletion/geneticsABSTRACT
Posttranslational protein translocation across the endoplasmic reticulum membrane of yeast requires a seven-component transmembrane complex (the Sec complex) in collaboration with the lumenal Kar2 protein (Kar2p). A translocation substrate was initially bound to the cytosolic face of the purified Sec complex in a signal-sequence-dependent but Kar2p- and nucleotide-independent manner. In a subsequent reaction, in which Kar2p interacted with the lumenal face of the Sec complex and hydrolyzed adenosine triphosphate, the substrate moved through a channel formed by the Sec complex and was released at the lumenal end. Movement through the channel occurred in detergent solution in the absence of a lipid bilayer.
Subject(s)
Fungal Proteins/metabolism , HSP70 Heat-Shock Proteins/metabolism , Heat-Shock Proteins , Membrane Proteins/metabolism , Membrane Transport Proteins , Protein Precursors/metabolism , Saccharomyces cerevisiae Proteins , Adenosine Triphosphate/metabolism , Biological Transport , Cross-Linking Reagents , Cytosol/metabolism , Detergents , Digitonin , Endoplasmic Reticulum/metabolism , Lipid Bilayers , Liposomes/metabolism , Protein Sorting Signals/metabolism , Proteolipids/metabolism , RNA, Transfer/metabolism , SEC Translocation Channels , Solubility , SuccinimidesABSTRACT
The heterotrimeric Sec61p complex is a major component of the protein-conducting channel of the endoplasmic reticulum (ER) membrane, associating with either ribosomes or the Sec62/63 complex to perform co- and posttranslational transport, respectively. We show by electron microscopy that purified mammalian and yeast Sec61p complexes in detergent form cylindrical oligomers with a diameter of approximately 85 A and a central pore of approximately 20 A. Each oligomer contains 3-4 heterotrimers. Similar ring structures are seen in reconstituted proteoliposomes and native membranes. Oligomer formation by the reconstituted Sec61p complex is stimulated by its association with ribosomes or the Sec62/63p complex. We propose that these cylindrical oligomers represent protein-conducting channels of the ER, formed by ligands specific for co- and posttranslational transport.
Subject(s)
Endoplasmic Reticulum/ultrastructure , Heat-Shock Proteins , Ion Channels/ultrastructure , Membrane Proteins/ultrastructure , Membrane Transport Proteins , Proteolipids/ultrastructure , Saccharomyces cerevisiae Proteins , Animals , Biological Transport , Cell Compartmentation , Detergents , Dogs , Freeze Fracturing , Fungal Proteins/metabolism , Image Enhancement , Ion Channel Gating , Macromolecular Substances , Membrane Proteins/isolation & purification , Membrane Proteins/metabolism , Models, Biological , Molecular Weight , Motion , Negative Staining , Particle Size , Protein Binding , Protein Biosynthesis , Protein Conformation , Ribosomes/metabolism , SEC Translocation Channels , YeastsABSTRACT
We have used proteolysis to examine the environment through which nascent secretory proteins are translocated across the membrane of the endoplasmic reticulum. After solubilization of rough microsomes with detergent, fragments comprised of the approximately 70 carboxyl-terminal amino acids of translocating nascent chains initiated and targeted in vivo were protected from digestion by added proteases. About 40 amino acids of nascent chains were protected from proteolysis by the ribosome; thus, membrane-derived components protect an additional 30 amino acids. Under conditions in which those 30 additional amino acids are protected, only a small set of integral membrane proteins remained associated with the ribosome. These proteins include the Sec61 complex previously identified as the core component of the membrane-bound protein translocation apparatus. These results support the concept of a translocation pore that makes intimate contact with the ribosome and thereby protects nascent chains from proteolytic digestion for an additional, constant length.