PubMed Health⌕ Search

Biomedical subjects

K Köhrer

Publications and source records attributed to K Köhrer.

43 records · Page 3Linked to original sources
PubMed1996

Multilamellar endosome-like compartment accumulates in the yeast vps28 vacuolar protein sorting mutant.

In the yeast Saccharomyces cerevisiae, vacuolar proteins such as carboxypeptidase Y transit from the Golgi to the lysosome-like vacuole via an endosome-like intermediate compartment. The vacuolar protein sorting (vps) mutant vps28, a member of the "class E" vps mutants, accumulates vacuolar, endocytic, and late Golgi markers in an aberrant endosome-like class E compartment. Sequence analysis of VPS28 revealed an open reading frame predicted to encode a hydrophilic protein of 242 amino acids. Consistent with this, polyclonal antiserum raised against Vps28p recognized a cytoplasmic protein of 28 kDa. Disruption of VPS28 resulted in moderate defects in both biosynthetic traffic and endocytic traffic destined for the vacuole. The transport of soluble vacuolar hydrolases to the vacuole was impaired in vps28 null mutant cells (approximately 40-50% carboxypeptidase Y missorted). Internalization of the endocytic marker FM 4-64, a vital lipophilic dye, resulted in intense staining of a small intracellular compartment adjacent to an enlarged vacuole in delta vps28 cells. Furthermore, the vacuolar H+-ATPase accumulated in the perivacuolar class E compartment in delta vps28 cells, as did a-factor receptor Ste3p that was internalized from the plasma membrane. Electron microscopic analysis revealed the presence of a novel compartment consisting of stacks of curved membrane cisternae. Immunolocalization studies demonstrated that the vacuolar H+-ATPase is associated with this cupped cisternal structure, indicating that it corresponds to the class E compartment observed by fluorescence microscopy. Our data indicate that kinetic defects in both anterograde and retrograde transport out of the prevacuolar compartment in vps28 mutants result in the accumulation of protein and membrane in an exaggerated multilamellar endosomal compartment. We propose that Vps28p, as well as other class E Vps proteins, may facilitate (possibly as coat proteins) the formation of transport intermediates required for efficient transport out of the prevacuolar endosome.

Amino Acid Sequence↗
PubMed1993

The yeast VPS17 gene encodes a membrane-associated protein required for the sorting of soluble vacuolar hydrolases.

vps17 mutants missort and secrete several vacuolar hydrolases. To analyze the role of the VPS17 gene in vacuolar protein delivery, we have cloned this gene by complementation of the vacuolar protein sorting defects of a vps17-5 mutant. Disruption of the VPS17 gene had no effect on the viability of haploid yeast cells, although they show an obvious defect in vacuolar morphology. vps17-disrupted cells contain numerous small vacuole-like compartments and also exhibit a severe defect in the sorting of carboxypeptidase Y (CPY), a soluble vacuolar hydrolase. 95% of CPY is missorted and secreted from the mutant cells. Vacuolar sorting of two other soluble hydrolases, proteinase A and proteinase B, is also affected, but to a lesser extent. Delivery and maturation of the vacuolar membrane protein alkaline phosphatase does not appear to be affected in a delta vps17 strain. The DNA sequence of the VPS17 clone indicates that the gene encodes a 551-amino-acid protein with a calculated molecular mass of 63.1 kDa. The protein sequence is hydrophilic and contains no obvious N-terminal signal sequence or hydrophobic membrane-spanning domains, indicating that the Vps17p does not enter the secretory pathway. Using a Vps17p-specific polyclonal antiserum, we have demonstrated that the Vps17 protein is not modified with N-linked carbohydrates at any of its four potential N-linked glycosylation sites. The Vps17 protein, however, fractionates to a particulate fraction after centrifugation at 100,000 x g. Vps17p can be released from this particulate fraction by treatment with either Triton X-100 or urea, indicating that the Vps17p is peripherally associated with a crude membrane fraction. Based on these results, we propose that the Vps17p functions on the cytoplasmic surface of some intracellular organelle, possibly the Golgi complex or an intermediate in Golgi to vacuole transport, to facilitate the sorting and delivery of soluble vacuolar hydrolases. Vacuolar membrane protein traffic, however, appears to occur by a mechanism that is independent of Vps17p function.

Amino Acid Sequence↗
PubMed1990

A yeast tRNA precursor containing a pre-mRNA intron is spliced via the pre-mRNA splicing mechanism.

We have replaced the 14 nucleotide long intervening sequence of the Saccharomyces cerevisiae SUP6 (ochre) tRNA(Tyr) gene by the 52 nucleotide long second intron of the S. cerevisiae MATa1 gene. Yeast cells containing this modified pre-tRNA showed the typical suppressor phenotype indicating that the MATa1 pre-mRNA intron was exactly excised in vivo from the primary tRNA transcript and a mature and functional tRNA was formed. Several lines of evidence show that the splicing reaction proceeded via the pre-mRNA splicing mechanism: the reaction yielded a lariat shaped excised intron with a lariat shaped intron-exon 2 molecule as intermediate; point mutations in the conserved UAC-UAAC box of the intron impaired splicing of the precursor RNA; in a temperature sensitive rna2 strain splicing of this tRNA precursor was inhibited at the restrictive temperature. Our results imply that in yeast the excision of a pre-mRNA intron is not dependent on the transcription apparatus by which it was generated and that transcription and splicing are uncoupled processes in vivo, too. Furthermore these data demonstrate that recognition of an RNA as a substrate for a pre-mRNA splicing reaction is, at least qualitatively, only intron dependent.

Base Sequence↗
PubMed1989

Specific oligodeoxynucleotide probes obtained through RNA sequencing.

By combining several established techniques we developed a method to test the specificity of mixed oligodeoxynucleotide hybridization probes and to provide the information for the design of long nondegenerate, and therefore more specific probes. Mixed oligodeoxynucleotide probes derived from known peptide sequences are first used to initiate primer extension reactions with poly(A)+RNA as template in the presence of three dNTPs and one ddNTP to generate cDNA transcripts of defined lengths. Comparing the lengths of the cDNA transcripts with the possible nucleic acid sequence coding for the known oligopeptide indicates whether the oligodeoxynucleotide mix hybridizes predominantly to the RNA of interest. In a second step, the oligodeoxynucleotide mix with the highest specificity is used for indirect RNA sequence analysis. This confirms the specificity of the probe and provides information to design a long, highly specific oligodeoxynucleotide probe for the gene of interest. This simple two-step-procedure helps to circumvent the time-consuming procedures of subcloning and sequencing of cross-hybridizing fragments.

Base Sequence↗
PubMed1988

Splicing and spliceosome formation of the yeast MATa1 transcript require a minimum distance from the 5' splice site to the internal branch acceptor site.

Small deletions of 6, 7, and 12 nucleotides introduced between the 5' splice site and the internal branch acceptor site of the first intron of the yeast MATa1 gene completely abolish accurate splicing in vitro in these constructs. Splicing only occurs at an alternative 5' splice site which was found in the first exon of the MATa1 gene and which is used both in vivo and in vitro. The splicing defect cannot be cured by expanding the distance from the branch point to the 3' splice site. If the alternative 5' splice site is deleted as well in these constructs, neither spliced products nor spliceosomes are formed. Our findings especially lead to the conclusion that a minimum distance between the 5' splice site and the internal branch acceptor site of the intron is required for the formation of splicing complexes and for accurate splicing.

Base Sequence↗
PubMed1985

Functional RNA polymerase II promoters in solitary retroviral long terminal repeats (LTR-IS elements).

LTR-IS elements are middle repetitive sequences in the mouse genome with structural features of solitary retroviral LTRs. In order to get some insight in the possible functional role of these sequences the promotor activity of two LTR-IS representatives differing by 105 bp in their U3 region was investigated. Gene fusions between LTR-IS sequences and the bacterial gene coding for chloramphenicol acetyl transferase (CAT) were transfected into mouse 3T6 cells and the expression of CAT was measured. It is shown that the LTR-IS sequences represent weak RNA polymerase II promoters which require enhancement by cis-or trans-activating factors.

Acetyltransferases↗