Structural and functional aspects of the multiplicity of Neu differentiation factors

We used molecular cloning and functional analyses to extend the family of Neu differentiation factors (NDFs) and to explore the biochemical activity of different NDF isoforms. Exhaustive cloning revealed the existence of six distinct fibroblastic pro-NDFs, whose basic transmembrane structure includes an immunoglobulin-like motif and an epidermal growth factor (EGF)-like domain. Structural variation is confined to three domains: the C-terminal portion of the EGF-like domain (isoforms alpha and beta), the adjacent juxtamembrane stretch (isoforms 1 to 4), and the variable-length cytoplasmic domain (isoforms a, b, and c). Only certain combinations of the variable domains exist, and they display partial tissue specificity in their expression: pro-NDF-alpha 2 is the predominant form in mesenchymal cells, whereas pro-NDF-beta 1 is the major neuronal isoform. Only the transmembrane isoforms were glycosylated and secreted as biologically active 44-kDa glycoproteins, implying that the transmembrane domain functions as an internal signal peptide. Extensive glycosylation precedes proteolytic cleavage of pro-NDF but has no effect on receptor binding. By contrast, the EGF-like domain fully retains receptor binding activity when expressed separately, but its beta-type C terminus displays higher affinity than alpha-type NDFs. Likewise, structural heterogeneity of the cytoplasmic tails may determine isoform-specific rate of pro-NDF processing. Taken together, these results suggest that different NDF isoforms are generated by alternative splicing and perform distinct tissue-specific functions.

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Membrane Orientation of the Human Papillomavirus Type 16 E5 Oncoprotein

Journal of Virology ◽

10.1128/jvi.01968-09 ◽

2009 ◽

Vol 84 (4) ◽

pp. 1696-1703 ◽

Cited By ~ 36

Author(s):

Ewa Krawczyk ◽

Frank A. Suprynowicz ◽

Sawali R. Sudarshan ◽

Richard Schlegel

Keyword(s):

Human Papillomavirus ◽

Transmembrane Domain ◽

Transmembrane Protein ◽

Biologically Active ◽

Human Papillomavirus Type 16 ◽

Cytoplasmic Proteins ◽

C Terminus ◽

Low Concentrations ◽

E5 Protein ◽

E5 Oncoprotein

ABSTRACT The E5 protein of human papillomavirus type 16 is a small, hydrophobic protein that localizes predominantly to membranes of the endoplasmic reticulum (ER). To define the orientation of E5 in these membranes, we employed a differential, detergent permeabilization technique that makes use of the ability of low concentrations of digitonin to selectively permeabilize the plasma membrane and saponin to permeabilize all cellular membranes. We then generated a biologically active E5 protein that was epitope tagged at both its N and C termini and determined the accessibility of these termini to antibodies in the presence and absence of detergents. In both COS cells and human ectocervical cells, the C terminus of E5 was exposed to the cytoplasm, whereas the N terminus was restricted to the lumen of the ER. Finally, the deletion of the E5 third transmembrane domain (and terminal hydrophilic amino acids) resulted in a protein with its C terminus in the ER lumen. Taken together, these topology findings are compatible with a model of E5 being a 3-pass transmembrane protein and with studies demonstrating its C terminus interacting with cytoplasmic proteins.

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Characterization of KIAA1427 protein as an atypical synaptotagmin (Syt XIII)

Biochemical Journal ◽

10.1042/bj3540249 ◽

2001 ◽

Vol 354 (2) ◽

pp. 249-257 ◽

Cited By ~ 12

Author(s):

Mitsunori FUKUDA ◽

Katsuhiko MIKOSHIBA

Keyword(s):

Transmembrane Domain ◽

Amino Acid Level ◽

Subcellular Fractionation ◽

Binding Activity ◽

Type I ◽

C2 Domains ◽

C Terminus ◽

Common Receptor ◽

Antibody Uptake

Synaptotagmin (Syt) belongs to a family of type-I membrane proteins and is a protein that consists of a short extracellular N-terminus, a single transmembrane domain, two C2 domains and a short C-terminus. Here, we cloned and characterized a mouse orthologue of human KIAA1427 protein as an atypical Syt (named Syt XIII). Subcellular fractionation and antibody-uptake experiments indicate that Syt XIII is indeed a type-I membrane protein, but, unlike other Syt isoforms, lacks an N-terminal extracellular domain. Syt XIII C2 domains show relatively little similarity to Syt I (less than 35% identity at the amino acid level), and lack key amino acids responsible for Ca2+ binding. Because of these substitutions, the Syt XIII C2 domains did not show Ca2+-dependent phospholipid-binding activity, and Syt XIII is thus classified as a Ca2+-independent isoform. By contrast, the Syt XIII C-terminal domain is highly homologous with other Syt isoforms and can function as a common receptor for neurexin Iα in vitro. Since Syt XIII is expressed in various tissues outside the brain, Syt XIII may be involved in constitutive vesicle transport.

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C-Terminal Deletions Can Suppress Temperature-Sensitive Mutations and Change Dominance in the Phage Mu Repressor

Genetics ◽

10.1093/genetics/142.3.661 ◽

1996 ◽

Vol 142 (3) ◽

pp. 661-672 ◽

Cited By ~ 5

Author(s):

Jodi L Vogel ◽

Vincent Geuskens ◽

Lucie Desmet ◽

N Patrick Higgins ◽

Ariane Toussaint

Keyword(s):

Binding Activity ◽

Temperature Sensitive ◽

Dna Binding Activity ◽

Heat Stable ◽

C Terminus ◽

Acid Domain ◽

Mutant Forms ◽

Amino Acid Domain

Abstract Mutations in an N-terminal 70-amino acid domain of bacteriophage Mu's repressor cause temperature-sensitive DNA-binding activity. Surprisingly, amber mutations can conditionally correct the heat-sensitive defect in three mutant forms of the repressor gene, cts25 (D43-G), cts62 (R47-Q and cts71 (M28-I), and in the appropriate bacterial host produce a heat-stable Sts phenotype (for survival of temperature shifts). Sts repressor mutants are heat sensitive when in supE or supF hosts and heat resistant when in Sup° hosts. Mutants with an Sts phenotype have amber mutations at one of three codons, Q179, Q187, or Q190. The Sts phenotype relates to the repressor size: in Sup° hosts sts repressors are shorter by seven, 10, or 18 amino acids compared to repressors in supE or supF hosts. The truncated form of the sts62-1 repressor, which lacks 18 residues (Q179–V196), binds Mu operator DNA more stably at 42° in vitro compared to its full-length counterpart (cts62 repressor). In addition to influencing temperature sensitivity, the C-terminus appears to control the susceptibility to in vivo Clp proteolysis by influencing the multimeric structure of repressor.

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