Biomedical subjects

Richard Zimmermann

Publications and source records attributed to Richard Zimmermann.

15 recordsLinked to original sources

PubMed2006

Protein transport into the endoplasmic reticulum: mechanisms and pathologies.

Transport into the endoplasmic reticulum (ER) is the crucial step in the biosynthesis of most secretory proteins and many membrane proteins. The products of the SIL1, SEC62 and SEC63 genes act in concert with the SEC61 complex and the molecular chaperones BiP and GRP170 to transport proteins into the ER. Interestingly, recent genetic work has linked mutations in the human and murine SIL1 genes to neurodegeneration, and mutations in the human SEC63 gene to autosomal dominant polycystic liver disease. Furthermore, mutations in the SEC63 gene and overexpression of the SEC62 gene are associated with various human cancers. Therefore, we suggest that these diseases should be considered to be pathologies of protein transport into the ER rather than protein-folding diseases.

Animals↗

PubMed2006

The nucleotide exchange factor activity of Grp170 may explain the non-lethal phenotype of loss of Sil1 function in man and mouse.

Recent genetic work characterized homozygous mutations in the SIL1 gene as cause for the neurodegeneration that is associated with Marinesco-Sjögren syndrome in man and the woozy mouse mutant. All reported mutations were expected to result in loss of Sil1 function. Sil1 has previously been shown to act as nucleotide exchange factor for the molecular chaperone immunoglobulin heavy chain binding protein (BiP) in the lumen of the endoplasmic reticulum (ER). In the yeast ER Lhs1p was shown to be able to substitute for Sil1p and to represent an alternative nucleotide exchange activity. Therefore, by analogy the mammalian ortholog of Lhs1p, Grp170, was suggested to be able to compensate for the loss of Sil1 function in many mammalian organs. Here we characterize mammalian Grp170 as alternative nucleotide exchange factor for BiP, thus providing a likely explanation for the non-lethal phenotype of the homozygous human and murine SIL1 mutations.

Animals↗

PubMed2006

Genomic and expression analysis of the 3q25-q26 amplification unit reveals TLOC1/SEC62 as a probable target gene in prostate cancer.

Gain at chromosome 3q25-q26 has been reported to commonly occur in prostate cancer. To map the 3q25-q26 amplification unit and to identify the candidate genes of amplification, we did fluorescence in situ hybridization and quantitative real-time PCR for gene copy number and mRNA expression measurements in prostate cancer cell lines and prostate cancer samples from radical prostatectomy specimens. The minimal overlapping region of DNA copy number gains in the cell lines could be narrowed down to 700 kb at 3q26.2. Of all positional and functional candidates in this region, the gene TLOC1/SEC62 revealed the highest frequency (50%) of copy number gains in the prostate cancer samples and was found to be up-regulated at the mRNA level in all samples analyzed. TLOC1/Sec62 protein was also shown to be overexpressed by Western blot analysis. Intriguingly, the TLOC1/SEC62 gene copy number was increased in prostate tumors from patients who had a lower risk of and a longer time to progression following radical prostatectomy. These findings make TLOC1/SEC62 the best candidate within the 3q amplification unit in prostate cancer. TLOC1/Sec62 protein is a component of the endoplasmic reticulum protein translocation machinery, whose function during prostate carcinogenesis remains to be determined.

Cell Line, Tumor↗

PubMed2005

Specific detection and semi-quantitative analysis of TRPC4 protein expression by antibodies.

In mouse tissues two variants of the transient receptor potential (canonical) (TRPC) 4 protein are expressed: the "full-length" TRPC4 protein and a slightly smaller variant, called TRPC4Delta(761-864), which lacks 84 amino acid residues. Although the presence of mRNA encoding the TRPC4 protein in mammalian cells and the detection of the heterologously expressed TRPC4 protein by Western blot analysis have been reported, the unequivocal detection of endogenous TRPC4 proteins has proven difficult. In the present study we compared polyclonal antibodies for the detection of TRPC4 proteins in mouse tissues and monitored their specificity and reliability by analysing corresponding tissues from TRPC4-deficient mice. In addition we introduced a procedure that allows us to estimate the amount of TRPC4 protein expressed in a single cell. Using this technique it appears that the amount of TRPC4 protein expressed stably in HEK 293 cells is at least fourfold higher than the amount of TRPC4 protein expressed endogenously in the bovine adrenocortical cell line SBAC.

Animals↗

PubMed2005

ERj1p has a basic role in protein biogenesis at the endoplasmic reticulum.

ERj1p is a membrane protein of the endoplasmic reticulum (ER) that can recruit the ER lumenal chaperone BiP to translating ribosomes. ERj1p can also modulate protein synthesis at initiation and is predicted to be a membrane-tethered transcription factor. Here we attribute the various functions of ERj1p to distinct regions within its cytosolic domain. A highly positively charged nonapeptide within this domain is necessary and sufficient for binding to ribosomes. Binding of ERj1p to ribosomes involves the 28S ribosomal RNA and occurs at the tunnel exit. Additionally, ERj1p has a dual regulatory role in gene expression: ERj1p inhibits translation in the absence of BiP, and another charged oligopeptide within the cytosolic domain of ERj1p mediates binding of the nuclear import factor importin beta and import into the nucleus, thereby paving the way for subsequent action on genomic DNA.

Animals↗

PubMed2005

ERj1p uses a universal ribosomal adaptor site to coordinate the 80S ribosome at the membrane.

Ribosomes translating secretory and membrane proteins are targeted to the endoplasmic reticulum membrane and attach to the protein-conducting channel and ribosome-associated membrane proteins (RAMPs). Recently, a new RAMP, ERj1p, has been identified that recruits BiP to ribosomes and regulates translational activity. Here we present the cryo-EM structure of a ribosome-ERj1p complex, revealing how ERj1p coordinates the ribosome at the membrane and how allosteric effects may mediate ERj1p's regulatory activity.

Animals↗

PubMed2005

Not all J domains are created equal: implications for the specificity of Hsp40-Hsp70 interactions.

Heat shock protein 40s (Hsp40s) and heat shock protein 70s (Hsp70s) form chaperone partnerships that are key components of cellular chaperone networks involved in facilitating the correct folding of a broad range of client proteins. While the Hsp40 family of proteins is highly diverse with multiple forms occurring in any particular cell or compartment, all its members are characterized by a J domain that directs their interaction with a partner Hsp70. Specific Hsp40-Hsp70 chaperone partnerships have been identified that are dedicated to the correct folding of distinct subsets of client proteins. The elucidation of the mechanism by which these specific Hsp40-Hsp70 partnerships are formed will greatly enhance our understanding of the way in which chaperone pathways are integrated into finely regulated protein folding networks. From in silico analyses, domain swapping and rational protein engineering experiments, evidence has accumulated that indicates that J domains contain key specificity determinants. This review will critically discuss the current understanding of the structural features of J domains that determine the specificity of interaction between Hsp40 proteins and their partner Hsp70s. We also propose a model in which the J domain is able to integrate specificity and chaperone activity.

Adenosine Triphosphatases↗

PubMed2004

Protein transport into canine pancreatic microsomes: a quantitative approach.

Transport of presecretory proteins into the mammalian rough endoplasmic reticulum involves a protein translocase that comprises the integral membrane proteins Sec61alphap, Sec61betap, and Sec61gammap as core components. Electron microscopic analysis of protein translocase in rough microsomal membranes suggested that between three and four heterotrimeric Sec61p complexes form the central unit of protein translocase. Here we analyzed the stoichiometry of heterotrimeric Sec61p complexes present in cotranslationally active protein translocases of canine pancreatic microsomes and various other lumenal and membrane components believed to be subunits of protein translocase and to be involved in covalent modifications. Based on these numbers, the capacity for cotranslational transport was estimated for the endoplasmic reticulum of the human pancreas.

Animals↗

PubMed2004

Characterization of pancreatic ERj3p, a homolog of yeast DnaJ-like protein Scj1p.

We have previously identified in the human EST sequence data base four overlapping clones that could be aligned with both a predicted protein sequence, deduced from the C. elegans genomic sequence, and partial amino acid sequences, obtained for a protein from canine pancreatic microsomes. We suggested that these proteins are homologs of yeast microsomal and DnaJ-like protein Scj1p and termed them ERj3p. Here we verified the predicted protein sequence of human ERj3p by sequence analysis of the corresponding cDNA. Multiple alignment of related sequences identified these proteins as true homologs of yeast Scj1p. Biochemical analysis of the canine protein characterized ERj3p as a soluble glycoprotein of the pancreatic endoplasmic reticulum. This pancreatic DnaJ-like protein was shown to interact with lumenal DnaK-like proteins, such as BiP. Furthermore, we found that ERj3p interacts with SDF2L1 protein that may be involved in protein O-glycosylation. We propose that ERj3p represents a cochaperone of DnaK-like chaperones of the mammalian endoplasmic reticulum and is involved in folding and maturation of newly synthesized proteins.

Amino Acid Sequence↗

PubMed2003

The Sec61p complex is a dynamic precursor activated channel.

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.

ADP Ribose Transferases↗

PubMed2003

Polypeptide-binding proteins mediate completion of co-translational protein translocation into the mammalian endoplasmic reticulum.

The first step in the secretion of most mammalian proteins is their transport into the lumen of the endoplasmic reticulum (ER). Transport of pre-secretory proteins into the mammalian ER requires signal peptides in the precursor proteins and a protein translocase in the ER membrane. In addition, hitherto unidentified lumenal ER proteins have been shown to be required for vectorial protein translocation. This requirement was confirmed in this study by using proteoliposomes that were made from microsomal detergent extracts and contained either low or high concentrations of lumenal ER proteins. Furthermore, immunoglobulin-heavy-chain-binding protein (BiP) was shown to be able to substitute for the full set of lumenal proteins and, in the case of biotinylated precursor proteins, avidin was found to be able to substitute for lumenal proteins. Thus, the polypeptide-chain-binding protein BiP was identified as one lumenal protein that is involved in efficient vectorial protein translocation into the mammalian ER.

Animals↗

PubMed2003

Transport of the intracisternal A-type particle Gag polyprotein to the endoplasmic reticulum is mediated by the signal recognition particle.

Intracisternal A-type particles (IAP) are defective endogenous retroviruses that accumulate in the endoplasmic reticulum (ER) of rodent cells. The enveloped particles are produced by assembly and budding of IAP Gag polyproteins at the ER membrane. In this study, we analyzed the specific ER transport of the Gag polyprotein of the IAP element MIA14. To this end, we performed in vitro translation of Gag in the presence of microsomal membranes or synthetic proteoliposomes followed by membrane sedimentation or flotation. ER binding of IAP Gag occurred mostly cotranslationally, and Gag polyproteins interacted specifically with proteoliposomes containing only signal recognition particle (SRP) receptor and the Sec61p complex, which form the minimal ER translocation apparatus. The direct participation of SRP in ER targeting of IAP Gag was demonstrated in cross-linking and immunoprecipitation experiments. The IAP polyprotein was not translocated into the ER; it was found to be tightly associated with the cytoplasmic side of the ER membrane but did not behave as an integral membrane protein. Substituting the functional signal peptide of preprolactin for the hydrophobic sequence at the N terminus of IAP Gag also did not result in translocation of the chimeric protein into the ER lumen, and grafting the IAP hydrophobic sequence onto preprolactin failed to yield luminal transport as well. These results suggest that the N-terminal hydrophobic region of the IAP Gag polyprotein functions as a transport signal which mediates SRP-dependent ER targeting, but polyprotein translocation or integration into the membrane is prevented by the signal sequence itself and by additional regions of Gag.

Amino Acid Sequence↗

PubMed2002

Tetratricopeptide repeat motif-mediated Hsc70-mSTI1 interaction. Molecular characterization of the critical contacts for successful binding and specificity.

Murine stress-inducible protein 1 (mSTI1) is a co-chaperone that is homologous with the human Hsp70/Hsp90-organizing protein (Hop). Guided by Hop structural data and sequence alignment analyses, we have used site-directed mutagenesis, co-precipitation assays, circular dichroism spectroscopy, steady-state fluorescence, and surface plasmon resonance spectroscopy to both qualitatively and quantitatively characterize the contacts necessary for the N-terminal tetratricopeptide repeat domain (TPR1) of mSTI1 to bind to heat shock cognate protein 70 (Hsc70) and to discriminate between Hsc70 and Hsp90. We have shown that substitutions in the first TPR motif of Lys(8) or Asn(12) did not affect binding of mSTI1 to Hsc70, whereas double substitution of these residues abrogated binding. A substitution in the second TPR motif of Asn(43) lowered but did not abrogate binding. Similarly, a deletion in the second TPR motif coupled with a substitution of Lys(8) or Asn(12) reduced but did not abrogate binding. These results suggest that mSTI1-Hsc70 interaction requires a network of interactions not only between charged residues in the TPR1 domain of mSTI1 and the EEVD motif of Hsc70 but also outside the TPR domain. We propose that the electrostatic interactions in the first TPR motif made by Lys(8) or Asn(12) define part of the minimum interactions required for successful mSTI1-Hsc70 interaction. Using a truncated derivative of mSTI1 incapable of binding to Hsp90, we substituted residues on TPR1 potentially involved in hydrophobic contacts with Hsc70. The modified protein had reduced binding to Hsc70 but now showed significant binding capacity for Hsp90. In contrast, topologically equivalent substitutions on a truncated derivative of mSTI1 incapable of binding to Hsc70 did not confer Hsc70 specificity on TPR2A. Our results suggest that binding of Hsc70 to TPR1 is more specific than binding of Hsp90 to TPR2A with serious implications for the mechanisms of mSTI1 interactions with Hsc70 and Hsp90 in vivo.

3T3 Cells↗

PubMed2002

Tail-anchored and signal-anchored proteins utilize overlapping pathways during membrane insertion.

Tail-anchored proteins are a distinct class of membrane proteins that are characterized by a C-terminal membrane insertion sequence and a capacity for post-translational integration. Although it is now clear that tail-anchored proteins are inserted into the membrane at the endoplasmic reticulum (ER), the molecular basis for their integration is poorly understood. We have used a cross-linking approach to identify ER components that may be involved in the membrane insertion of tail-anchored proteins. We find that several newly synthesized tail-anchored proteins are transiently associated with a defined subset of cellular components. Among these, we identify several ER proteins, including subunits of the Sec61 translocon, Sec62p, Sec63p, and the 25-kDa subunit of the signal peptidase complex. When we analyze the cotranslational membrane insertion of a comparable signal-anchored protein we find the nascent polypeptide associated with a similar set of ER components. We conclude that the pathways for the integration of tail-anchored and signal-anchored membrane proteins at the ER exhibit a substantial degree of overlap, and we propose that this reflects similarities between co- and post-translational membrane insertion.

Cell Membrane↗

PubMed2002

A novel type of co-chaperone mediates transmembrane recruitment of DnaK-like chaperones to ribosomes.

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.

Amino Acid Sequence↗