Identification, developmental regulation, and response to heat shock of two antigenically related forms of a major nuclear envelope protein in Drosophila embryos: application of an improved method for affinity purification of antibodies using polypeptides immobilized on nitrocellulose blots

Smith, David Eugene; Fisher, Paul A.

doi:10.1083/jcb.99.1.20

Cited by 665 publications

(326 citation statements)

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“…The fusion product was purified under denaturing conditions (8 M urea) with the use of Ni-NTA agarose (Qiagen, Chatsworth, CA), and dialyzed into phosphate-buffered saline. The resulting serum was then immunoabsorbed against the SNG-1 fusion protein bound to nitrocellulose as described by Smith and Fisher (1984). Immunocytochemistry with H. Zhao and M.L.…”

Section: Immunocytochemistrymentioning

confidence: 99%

A Conserved Mechanism of Synaptogyrin Localization

Zhao

Nonet

2001

MBoC

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We have studied the localization of synaptogyrin family members in vivo. Both native and green fluorescent protein (GFP)-tagged Caenorhabditis elegans synaptogyrin (SNG-1) are expressed in neurons and synaptically localized. Deletion and mutational analysis with the use of GFP-tagged SNG-1 has defined a 38 amino acid sequence within the C terminus of SNG-1 and a single arginine in the cytoplasmic loop between transmembrane domain 2 and 3 that are required for SNG-1 localization. These domains may represent components of signals that target synaptogyrin for endocytosis from the plasma membrane and direct synaptogyrin to synaptic vesicles, respectively. In chimeric studies, these regions were sufficient to relocalize cellugyrin, a nonneuronal form of synaptogyrin, from nonsynaptic regions such as the sensory dendrites and the cell body to synaptic vesicles. Furthermore, GFP-tagged rat synaptogyrin is synaptically localized in neurons of C. elegans and in cultured hippocampal neurons. Similarly, the C-terminal domain of rat synaptogyrin is necessary for localization in hippocampal neurons. Our study suggests that the mechanisms for synaptogyrin localization are likely to be conserved from C. elegans to vertebrates. INTRODUCTIONSynaptic vesicles (SVs) contain a restricted set of membrane proteins important for neuronal function (Sü dhof, 1995;Calakos and Scheller, 1996;Fernandez-Chacon and Sü dhof, 1999). The mechanisms responsible for targeting these proteins specifically to the SV membrane are poorly understood. Integral membrane proteins are synthesized on the rough endoplasmic reticulum and traffic through the Golgi network. Proteins exiting the trans-Golgi network (TGN) are sorted to different types of transport vesicles. SV proteins are sorted to synaptic vesicle precursors (preSVs), which differ from mature SVs both in morphology and protein composition (Tsukita and Ishikawa, 1980; Okada et al., 1995). Evidence supports both direct routing of SV proteins from TGN to preSVs and indirect routing via the plasma membrane (reviewed by Hannah et al., 1999). preSVs are subsequently transported along axonal processes to the nerve terminal by motor proteins of the kinesin superfamily (reviewed by Hirokawa, 1998). How preSVs mature after delivery to the nerve terminal is unknown. preSVs might fuse with an endosomal compartment and SVs generated by budding from the endosome. A second, but not mutually exclusive possibility, is that preSVs are delivered to the presynaptic plasma membrane at the nerve terminal and then retrieved by the same mechanism(s) used to recycle SVs after regulated exocytosis (Hannah et al., 1999; reviewed by Buckley et al., 2000). Regardless of the exact route of trafficking to the mature organelle, SV proteins must be sorted away from other membrane proteins at several stages during their life cycle.Signal sequences resident in proteins are thought to mediate the sorting of many proteins to cellular compartments. Several studies have identified domains and motifs necessary for correct localizati...

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Section: Immunocytochemistrymentioning

confidence: 99%

A Conserved Mechanism of Synaptogyrin Localization

Zhao

Nonet

2001

MBoC

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“…All the specific mouse and rabbit antibodies obtained were affinity-purified using the electro-transferred overexpressed antigen according to the method of Smith and Fisher (1984). For protein gel blot analysis these antibody preparations were used in a 1:5 dilution.…”

Section: Expression Of Phytoene Desaturase In Insect Cells Antibody mentioning

confidence: 99%

A novel, soluble form of phytoene desaturase from Narcissus pseudonarcissus chromoplasts is Hsp70‐complexed and competent for flavinylation, membrane association and enzymatic activation

Al‐Babili

Lintig

Haubruck³

et al. 1996

The Plant Journal

112

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SummaryA cDNA coding for the carotenoid biosynthetic enzyme phytoene desaturase from Narcissus pseudonarcissus was cloned and the corresponding protein expressed in insect cells using the baculovirus system. Polyclonal antibodies raised against the recombinant protein allowed the detection of soluble and tightly membrane-bound populations of phytoene desaturase in the chromoplasts isolated from petals. The soluble form is enzymatically inactive and a constituent of a larger Hsp 7g-containing protein complex in the stroma, whereas the membrane-bound form is functional. In vitro, the soluble form is able to associate on to/into protein-free liposomal membranes made from chromoplast lipids, thereby gaining activity by binding added flavine adenine dinucleotide (FAD). Once bound to membranes, activated phytoene desaturase works independently of any added FAD, employing membrane-bound electron acceptors. FAD, however, exerts no positive effect on the membrane-association process, its role is confined to enzymatic activation. Although carotenoid accumulation is strongly induced during flower development, only very low concentrations of phytoene desaturase transcripts are detectable, while the corresponding protein accumulates in low, but measurable amounts, appearing in soluble and membrane-bound states. Poet-transcriptional mechanisms contribute significantly to carotenoid accumulation, as do factors determining the enzymatic activity of phytoene desaturase, for example by influencing the redox-state of membrane-bound electron acceptors.

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“…Specific antibodies against the FP were purified from immunoblots according to Smith and Fisher [20].…”

Section: Preparation Of Polyclonal Antibodies and Western Blottingmentioning

confidence: 99%

Acidic pH generated by H⁺‐ATPase pumps triggers the activity of a fusogenic protein associated with rat liver endoplasmic reticulum

Monni

Roberti

Corazzi

2001

European Journal of Biochemistry

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Fusogenic protein (FP) is a glycoprotein (< 50 kDa), previously purified by us from rat liver endoplasmic reticulum, which explicates fusogenic activity at acidic pH in vitro. To suggest a possible role of FP in membrane fusion, the topology of the protein in the membrane and the conditions in which FP is operating in microsomes have been investigated. Anti-FP polyclonal antibodies inhibited pure FP activity, but not the protein activity in microsomes, suggesting interaction of antibodies with a part of FP concealed in intact membranes. FP activity in microsomes was lost after treatment with Pronase. Western blot analysis of Pronase-treated microsomes showed that the proteolysis removed a fragment (< 5 kDa). This fragment is exposed on the outer surface of microsomes and involved in fusogenic activity, whereas the largest part of FP is embedded in microsomal vesicles. Therefore, FP can be affected by modifications on the cytosolic and luminal sides of microsomal membranes. Indeed, when microsomal lumen was acidified by H 1 -ATPase activity, binding and fusion of fluorescent labelled liposomes to microsomes occurred. Direct involvement of FP in the fusogenic event was observed by reconstituting pure FP in liposomes with a preformed H 1 gradient. FP triggered a fusion process in response to the acidic interior of liposomes, despite an exterior 7.4 pH unable to promote fusogenic protein activity. As intracellular membrane fusion occurs at neutral pH involving the cytosolic sides of membranes, FP may participate in this event by exploiting the acidic pH formed in the lumen of endoplasmic reticulum through H 1 -translocating ATPase activity.Keywords: acidic fusion; fusogenic protein; endoplasmic reticulum; membrane fusion; H 1 -ATPase.The endoplasmic reticulum is element of exo-and endocytic pathways of the vacuolar system that continuously undergoes fusion and fission [1]; these processes are mediated by protein factors [2,3]. Endosomal membrane fusion is regulated by GTP-binding proteins [4]. Annexin II, together with other proteins, is a major component of fusogenic endosomal vesicles [5]. The presence of membrane-bound proteins in cells that mediate membrane± liposome interaction by a fusion mechanism has been reported [6±8]. All these proteins explicate their activity at neutral pH. Alternatively, fusogenic proteins of viruses that enter the cell through receptor-mediated endocytosis share a common feature of being activated at a low pH in the endosomal compartment [9]. We have purified a fusogenic protein (FP) from rat liver endoplasmic reticulum which promotes aggregation and fusion of vesicles at acidic pH [10]. Considering the dynamic feature of the endoplasmic reticulum, the presence of FP in these membranes makes it a candidate for membrane fusion and worth being studied and characterized for a possible role in vivo.The endoplasmic reticulum is the main location of lipid biosynthetic enzymes [11] and source of intense importation of lipid for membrane assembly or phospholipid remodeling [12]. A mixed popul...

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Identification, developmental regulation, and response to heat shock of two antigenically related forms of a major nuclear envelope protein in Drosophila embryos: application of an improved method for affinity purification of antibodies using polypeptides immobilized on nitrocellulose blots

Cited by 665 publications

References 31 publications

A Conserved Mechanism of Synaptogyrin Localization

A Conserved Mechanism of Synaptogyrin Localization

A novel, soluble form of phytoene desaturase from Narcissus pseudonarcissus chromoplasts is Hsp70‐complexed and competent for flavinylation, membrane association and enzymatic activation

Acidic pH generated by H⁺‐ATPase pumps triggers the activity of a fusogenic protein associated with rat liver endoplasmic reticulum

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Identification, developmental regulation, and response to heat shock of two antigenically related forms of a major nuclear envelope protein in Drosophila embryos: application of an improved method for affinity purification of antibodies using polypeptides immobilized on nitrocellulose blots

Cited by 665 publications

References 31 publications

A Conserved Mechanism of Synaptogyrin Localization

A Conserved Mechanism of Synaptogyrin Localization

A novel, soluble form of phytoene desaturase from Narcissus pseudonarcissus chromoplasts is Hsp70‐complexed and competent for flavinylation, membrane association and enzymatic activation

Acidic pH generated by H+‐ATPase pumps triggers the activity of a fusogenic protein associated with rat liver endoplasmic reticulum

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Acidic pH generated by H⁺‐ATPase pumps triggers the activity of a fusogenic protein associated with rat liver endoplasmic reticulum