[2] Subcellular fractionation of rat liver

Fleischer, Sidney; Kervina, Marija

doi:10.1016/0076-6879(74)31005-1

Cited by 409 publications

(171 citation statements)

References 29 publications

Supporting

Mentioning

164

Contrasting

Unclassified

Order By: Relevance

“…Western Blotting Experiment-Subcellular fractionation of cells was performed by differential centrifugation as described previously (13). For each fraction, 20 g of protein were separated on 12.5% SDSpolyacrylamide gel electrophoresis and transferred to nitrocellulose for Western blotting.…”

Section: Methodsmentioning

confidence: 99%

The Human Testis Determining Factor SRY Binds a Nuclear Factor Containing PDZ Protein Interaction Domains

Poulat

Barbara

Desclozeaux

et al. 1997

Journal of Biological Chemistry

144

View full text Add to dashboard Cite

The human Y-linked testis determining gene SRY encodes a protein with a DNA binding domain from the high mobility group box family. To date, no function has been assigned to amino acid sequences located outside this DNA binding motif. Here, we identify in a yeast two-hybrid screen a PDZ protein termed SIP-1, as an interacting protein with human SRY. In vitro, biochemical analysis, immunoprecipitation experiments, as well as expression of SIP-1 in human embryonic testis confirm that the two proteins can interact together. Interacting domains were mapped to the C-terminal seven amino acids of SRY and to the PDZ domains of SIP-1, respectively. We hypothesize that SIP-1 could connect SRY to other transcription factors providing SRY for its missing trans-regulation domain.In mammals, male sex determination is controlled by genetic information encoded on the Y chromosome and leads to the differentiation of embryonic gonads into testes. SRY, a Y-specific gene cloned in 1990 (1), was shown to meet all of the criteria of the testis determining factor (2, 3). SRY encodes a small nuclear protein of 204 residues comprising three distinct domains, with a central domain of about 78 amino acids called the high mobility group (HMG) 1 box. This central domain includes a nuclear localization signal (4), and in vitro studies of the human SRY protein have demonstrated its sequence-specific DNA binding through this HMG box (5). To date, no function has been ascribed to the regions of the human SRY protein outside the HMG box, whereas in mouse Sry, the Cterminal part of the protein can function as a transcriptional activator (6). This apparent lack of human SRY transactivation domain and the background-dependent sex determination capabilities of certain mouse Y chromosomes (7) along with the growing list of HMG box containing proteins involved in the assembly of multiprotein complexes (8, 9) suggest that SRY might interact with other proteins. Indeed regulation of target genes by SRY may require collaboration with other factors interacting directly with the SRY protein. Here, using the two-hybrid system we show that a PDZ domain containing nuclear protein interacts with the C-terminal portion of nonrodent SRY. Our two-hybrid results were confirmed by in vitro experiments and by immunoprecipitation that demonstrate that SRY and SIP-1 are associated also in vivo. Finally, immunofluorescence experiments reveal a nuclear expression pattern for SIP-1 in a cell line as well as in human embryo cuts at the level of the genital ridges. These results suggest a model in which SIP-1 could permit to connect SRY to another protein harboring a PDZ binding motif. The nature of this other protein partner and the functional implication of such a complex in human sex determination are now under investigation. MATERIALS AND METHODSLibrary Screenings-Yeast two-hybrid screening was carried out using the HF7c yeast strain harboring HIS3 and ␤-gal reporter genes under the control of upstream GAL4-binding sites as originally described (10, 11). The bait cons...

show abstract

Section: Methodsmentioning

confidence: 99%

The Human Testis Determining Factor SRY Binds a Nuclear Factor Containing PDZ Protein Interaction Domains

Poulat

Barbara

Desclozeaux

et al. 1997

Journal of Biological Chemistry

144

View full text Add to dashboard Cite

show abstract

“…Subcellular fractions of mouse liver were obtained as described (11). Purity of the fractions was assessed by assays for DNA, citrate synthase (12), acid phosphatase (13), lactate dehydrogenase (14), Na ϩ /K ϩ ATPase (15), and glucose 6-phosphatase (16).…”

Section: Methodsmentioning

confidence: 99%

Metaxin Is a Component of a Preprotein Import Complex in the Outer Membrane of the Mammalian Mitochondrion

Armstrong

Komiya

Bergman

et al. 1997

Journal of Biological Chemistry

124

106

View full text Add to dashboard Cite

Metaxin, a novel gene located between the glucocerebrosidase and thrombospondin 3 genes in the mouse, is essential for survival of the postimplantation mouse embryo. In this study, the subcellular location, domain structure, and biochemical function of metaxin were investigated. Anti-recombinant metaxin antibodies recognized 35-and 70-kDa proteins in mitochondria from various tissues; the 35-kDa protein is consistent in size with the predicted translation product of metaxin cDNA. When metaxin cDNA was transfected into COS cells, immunofluorescence staining demonstrated that the protein is located in mitochondria. Metaxin contains a putative mitochondrial outer membrane signal anchor domain at its C terminus, and a truncated form of metaxin lacking this signal anchor domain had a reduced association with mitochondria. In addition, metaxin was highly susceptible to proteases in intact mitochondria. We therefore conclude that metaxin is a mitochondrial protein that extends into the cytosol while anchored into the outer membrane at its C terminus. In its N-terminal region, metaxin shows significant sequence identity to Tom37, a component of the outer membrane portion of the mitochondrial preprotein translocation apparatus in Saccharomyces cerevisiae, but important structural differences, including apparently different mechanisms of targeting to membranes, also exist between the two proteins. Given the similar subcellular locations of metaxin and Tom37, the possible role of metaxin in mitochondrial preprotein import was investigated. Antibodies against metaxin, when preincubated with mitochondria, partially inhibited the uptake of radiolabeled preadrenodoxin into mitochondria. Metaxin is therefore the second mammalian component of the protein translocation apparatus of the mitochondrial outer membrane to be characterized at the molecular level and the first for which an inherited mutation has been described. The early embryonic lethal phenotype of mice lacking metaxin demonstrates that efficient import of proteins into mitochondria is crucial for cellular survival. The characterization of metaxin provides an opportunity to elucidate similarities and possible differences in the mechanisms of protein import between fungi and mammals and in the phenotypes of fungi and mammals lacking mitochondrial import receptors.During studies of thrombospondin 3 (Thbs3), which encodes an extracellular matrix protein (2-4), and glucocerebrosidase (Gba), which encodes a lysosomal enzyme (5), a novel gene, termed metaxin (Mtx), was found to span the interval between Thbs3 and Gba on chromosome 3E3-F1 in the mouse (6). Mtx and Thbs3 are situated in a head-to-head orientation, with the translation start sites 1.4 kilobase pairs apart. Gba and Mtx lie in a tail-totail orientation, with the polyadenylation sites spaced 431 base pairs apart. The Mtx promoter resembles the promoters for housekeeping genes in that it is TATA-less, GC-rich, and regulated by tandem Sp1 elements within 300 base pairs of the transcription start site (7). Although ...

show abstract

“…The resulting supematant, Abbreviations: GMPCPP, guanylyl-(a,13)-methylene-diphosphonate; mem, membrane extract; MTP, microtubule protein; 1D-PAGE, one-dimensional PAGE. Jones and Matus (1974) and Fleischer and Kervina (1978) were combined and modified as follows. Liver from adult male Sprague Dawley rats was removed, washed twice with 10% sucrose, 10 mM N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), pH 7.4, and homogenized in 5 volumes of the sucrose-HEPES buffer.…”

Section: Purification Of Microtubule Proteinmentioning

confidence: 99%

Posttranslational modification of tubulin by palmitoylation: I. In vivo and cell-free studies.

Caron

1997

MBoC

View full text Add to dashboard Cite

It is well established that microtubules interact with intracellular membranes of eukaryotic cells. There is also evidence that tubulin, the major subunit of microtubules, associates directly with membranes. In many cases, this association between tubulin and membranes involves hydrophobic interactions. However, neither primary sequence nor known posttranslational modifications of tubulin can account for such an interaction. The goal of this study was to determine the molecular nature of hydrophobic interactions between tubulin and membranes. Specifically, I sought to identify a posttranslational modification of tubulin that is found in membrane proteins but not in cytoplasmic proteins. One such modification is the covalent attachment of the long chain fatty acid palmitate. The possibility that tubulin is a substrate for palmitoylation was investigated. First, I found that tubulin was palmitoylated in resting platelets and that the level of palmitoylation of tubulin decreased upon activation of platelets with thrombin. Second, to obtain quantities of palmitoylated tubulin required for protein structure analysis, a cell-free system for palmitoylation of tubulin was developed and characterized. The substrates for palmitoylation were nonpolymerized tubulin and tubulin in microtubules assembled with the slowly hydrolyzable GTP analogue guanylyl-(a,3)-methylenediphosphonate. However, tubulin in Taxol-assembled microtubules was not a substrate for palmitoylation. Likewise, palmitoylation of tubulin in the cell-free system was specifically inhibited by the antimicrotubule drugs Colcemid, podophyllotoxin, nocodazole, and vinblastine. These experiments identify a previously unknown posttranslational modification of tubulin that can account for at least one type of hydrophobic interaction with intracellular membranes.

show abstract

[2] Subcellular fractionation of rat liver

Cited by 409 publications

References 29 publications

The Human Testis Determining Factor SRY Binds a Nuclear Factor Containing PDZ Protein Interaction Domains

The Human Testis Determining Factor SRY Binds a Nuclear Factor Containing PDZ Protein Interaction Domains

Metaxin Is a Component of a Preprotein Import Complex in the Outer Membrane of the Mammalian Mitochondrion

Posttranslational modification of tubulin by palmitoylation: I. In vivo and cell-free studies.

Contact Info

Product

Resources

About