Biomedical subjects
Publications and source records attributed to S K Lyman.
In vivo studies on the dynamics of the nuclear pore complex (NPC) in yeast suggested that NPCs are highly mobile in the nuclear envelope. However, new evidence indicates that in mammalian cells NPCs are stably attached to a flexible lamina framework, but a peripheral component can exchange rapidly with an intranuclear pool.
We have identified an apparent human homolog of the yeast Nop5/Nop58 protein. hNop5/Nop58 codes for a protein of predicted molecular weight 59.6 kDa and is 46.8% identical to Saccharomyces cerevisiae Nop5/Nop58. Immunofluorescent staining with antibodies against hNop5/Nop58 indicate that it is localized primarily to the nucleolus, and coimmunoprecipitation from nuclear extracts demonstrates that hNop5/Nop58 interacts with the box C/D family of snoRNAs. Thus, hNop5/Nop58 is a common component of the box C/D snoRNPs, and joins fibrillarin as the second such component identified and characterized in metazoans.
The translocation of a secretory precursor protein across the ER membrane comprises three phases: docking of the precursor at the membrane, insertion into the translocation pore, and exit from the pore into the ER lumen. We demonstrate that Sec62p, Sec71p and Sec72p form a translocon subcomplex that engages secretory precursors at the membrane site of the ER translocation machinery. Binding of a precursor to the subcomplex depends on the presence of an intact signal sequence and occurs only in the absence of ATP. In the presence of ATP, the precursor is released from the subcomplex in a reaction mediated by the lumenal hsp70, BiP. This release reaction, which is specific to BiP and requires interaction between BiP and the DnaJ homolog Sec63p, defines a role for BiP and Sec63p early in the ER translocation process.
Proteins enter the secretory pathway by two general routes. In one, the complete polypeptide is made in the cytoplasm and held in an incompletely folded state by chaperoning adenosine triphosphatases (ATPases) such as hsp70. In Saccharomyces cerevisiae, fully synthesized secretory precursors engage the endoplasmic reticulum (ER) membrane by interaction with a set of Sec proteins comprising the polypeptide translocation apparatus (Sec61p, Sec62p, Sec63p, Sec71p, Sec72p). Productive interaction requires displacement of hsp70 from the precursor, a reaction that is facilitated by Ydj1p, a homologue of the Escherichia coli DnaJ protein. Both DnaJ and Ydj1p regulate chaperone activity by stimulating the ATPase activity of their respective hsp70 partners (E. coli DnaK and S. cerevisiae Ssa1p, respectively). In the ER lumen, another hsp70 chaperone, BiP, binds ATP and interacts with the ER membrane via its contact with a peptide loop of Sec63p. This loop represents yet another DnaJ homologue in that it contains a region of approximately 70 residue similarity to the 'J box', the most conserved region of the DnaJ family of proteins. In the presence of ATP, under conditions in which BiP can bind to Sec63p, the secretory precursor passes from the cytosol into the lumen through a membrane channel formed by Sec61p. A second route to the membrane pore that is used by many other secretory precursors, particularly in mammalian cells, requires that the polypeptide engage the ER membrane as the nascent chain emerges from the ribosome. Such cotranslational translocation bypasses the need for certain Sec proteins, instead utilizing an alternate set of cytosolic and membrane factors that allows the nascent chain to be inserted directly into the Sec61p channel.
To clarify the roles of Kar2p (BiP) and Sec63p in translocation across the ER membrane in Saccharomyces cerevisiae, we have utilized mutant alleles of the essential genes that encode these proteins: kar2-203 and sec63-1. Sanders et al. (Sanders, S. L., K. M. Whitfield, J. P. Vogel, M. D. Rose, and R. W. Schekman. 1992. Cell. 69:353-365) showed that the translocation defect of the kar2-203 mutant lies in the inability of the precursor protein to complete its transit across the membrane, suggesting that the lumenal hsp70 homologue Kar2p (BiP) binds the transiting polypeptide in order to facilitate its passage through the pore. We now show that mutation of a conserved residue (A181-->T) (Nelson, M. K., T. Kurihara, and P. Silver. 1993. Genetics. 134:159-173) in the lumenal DnaJ box of Sec63p (sec63-1) results in an in vitro phenotype that mimics the precursor stalling defect of kar2-203. We demonstrate by several criteria that this phenotype results specifically from a defect in the lumenal interaction between Sec63p and BiP: Neither a sec62-1 mutant nor a mutation in the cytosolically exposed domain of Sec63p causes precursor stalling, and interaction of the sec63-1 mutant with the membranebound components of the translocation apparatus is unimpaired. Additionally, dominant KAR2 suppressors of sec63-1 partially relieve the stalling defect. Thus, proper interaction between BiP and Sec63p is necessary to allow the precursor polypeptide to complete its transit across the membrane.
Initiation of DNA replication by phage lambda requires the ordered assembly and disassembly of a specialized nucleoprotein structure at the origin of replication. In the disassembly pathway, a set of Escherichia coli heat shock proteins termed the Hsp70 complex--DnaK, DnaJ, and GrpE--act with ATP to release lambda P protein from the nucleo-protein complex, freeing the DnaB helicase for its DNA-unwinding reaction. To investigate the mechanism of the release reaction, we have examined the interaction between P and the three heat shock proteins by glycerol gradient sedimentation and gel electrophoresis. We have discovered an ATP-dependent ternary interaction between P, DnaK, and DnaJ; this P.DnaK.DnaJ complex is dissociated by GrpE. We have concluded that the function of the Hsp70 complex in sequestering and releasing P protein provides for the critical step in the disassembly pathway. Based on our data and other work on protein folding, the formation of the P.DnaK.DnaJ complex might involve a conformational shift to a folding intermediate of P.