Anchor Signal Sequence (anchor + signal_sequence)

Distribution by Scientific Domains


Selected Abstracts


Mutagenesis studies in transgenic Xenopus intermediate pituitary cells reveal structural elements necessary for correct prion protein biosynthesis

DEVELOPMENTAL NEUROBIOLOGY, Issue 6 2007
Jos W.G. van Rosmalen
Abstract The cellular prion protein (PrPC) is generally accepted to be involved in the development of prion diseases, but its physiological role is still under debate. To obtain more insight into PrPC functioning, we here used stable Xenopus transgenesis in combination with the proopiomelanocortin (POMC) gene promoter to express mutated forms of Xenopus PrPC fused to the C-terminus of the green fluorescent protein (GFP) specifically in the neuroendocrine Xenopus intermediate pituitary melanotrope cells. Similar to GFP-PrPC, the newly synthesized GFP-PrPCK81A mutant protein was stepwise mono- and di-N-glycosylated to 48- and 51-kDa forms, respectively, and eventually complex glycosylated to yield a 55-kDa mature form. Unlike GFP-PrPC, the mature GFP-PrPCK81A mutant protein was not cleaved, demonstrating the endoproteolytic processing of Xenopus PrPC at lysine residue 81. Surprisingly, removal of the glycosylphosphatidylinositol (GPI) anchor signal sequence or insertion of an octarepeat still allowed N-linked glycosylation, but the GFP-PrPC,GPI and GFP-PrPCocta mutant proteins were not complex glycosylated and not cleaved, indicating that the GPI/octa mutants did not reach the mid-Golgi compartment of the secretory pathway. The transgene expression of the mutant proteins did not affect the ultrastructure of the melanotrope cells nor POMC biosynthesis and processing, or POMC-derived peptide secretion. Together, our findings reveal the evolutionary conservation of the site of metabolic cleavage and the importance of the presence of the GPI anchor and the absence of the octarepeat in Xenopus PrPC for its correct biosynthesis. 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2007. [source]


Hypoxia induces expression of a GPI-anchorless splice variant of the prion protein

FEBS JOURNAL, Issue 11 2008
Yutaka Kikuchi
The human prion protein (PrP) is a glycoprotein with a glycosylphosphatidylinositol (GPI) anchor at its C-terminus. Here we report alternative splicing within exon 2 of the PrP gene (PRNP) in the human glioblastoma cell line T98G. The open reading frame of the alternatively spliced mRNA lacked the GPI anchor signal sequence and encoded a 230 amino acid polypeptide. Its product, GPI-anchorless PrP (GPI, PrPSV), was unglycosylated and soluble in non-ionic detergent, and was found in the cytosolic fraction. We also detected low levels of alternatively spliced mRNA in human brain and non-neuronal tissues. When long-term passaged T98G cells were placed in a low-oxygen environment, alternatively spliced mRNA expression increased and expression of normally spliced PrP mRNA decreased. These findings imply that oxygen tension regulates GPI, PrPSV expression in T98G cells. [source]


Dual fluorescent protein reporters for studying cell behaviors in vivo

GENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 10 2009
M. David Stewart
HeLa cells transfected with plasmids expressing dual fluorescent protein reporters with cherry, green, and cyan fluorscent proteins. Histone 2B fusions localize fluorescent proteins to the nucleus, while fluorescent proteins fused to glycosylphosphatidylinositol anchor signal sequence localizes to plasma membranes. These dual fluorescent reporters label nuclear and plasma membranes of live cells to follow their behaviors in developing embryos. See the paper by Stewart et al. in this issue. [source]


Dual fluorescent protein reporters for studying cell behaviors in vivo

GENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 10 2009
M. David Stewart
Abstract Fluorescent proteins (FPs) are useful tools for visualizing live cells and their behaviors. Protein domains that mediate subcellular localization have been fused to FPs to highlight cellular structures. FPs fused with histone H2B incorporate into chromatin allowing visualization of nuclear events. FPs fused to a glycosylphosphatidylinositol anchor signal sequence label the plasma membrane, highlighting cellular shape. Thus, a reporter gene containing both types of FP fusions would allow for effective monitoring of cell shape, movement, mitotic stage, apoptosis, and other cellular activities. Here, we report a binary color-coding system using four differently colored FP reporters that generates 16 distinct color codes to label the nuclei and plasma membranes of live cells in culture and in transgenic mice. As an initial test of this system in vivo, the promoter of the human Ubiquitin C (UBC) gene was used to widely express one of the color-code reporters. Widespread expression of the reporter was attained in embryos; however, both male and female transgenic mice were infertile. In contrast, the promoter of the mouse Oct4/Pou5f1 gene linked to two different color-code reporters specifically labeled blastocysts, primordial germ cells, and postnatal germ cells, and these mice were fertile. Time-lapse movies of fluorescently-labeled primordial germs cells demonstrate the utility of the color-code system to visualize cell behaviors. This set of new FP reporters should be a useful tool for labeling distinct cell populations and studying their behaviors in complex tissues in vivo. genesis 47:708,717, 2009. 2009 Wiley-Liss, Inc. [source]