Transport route for synaptobrevin via a novel pathway of insertion into the endoplasmic reticulum membrane.

Kutay, Ulrike; Ahnert‐Hilger, Gudrun; Hartmann, Enno; Wiedenmann, Bertram; Rapoport, Tom A.

doi:10.1002/j.1460-2075.1995.tb06994.x

Cited by 280 publications

(317 citation statements)

References 44 publications

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“…membrane-water interface (Ridder et al, 2000). Because synaptobrevin and syntaxin represent a class of proteins called "tail-anchored" proteins that are inserted into the membrane posttranslationally (Kutay et al, 1993(Kutay et al, , 1995, it is conceivable that the membrane proximal region of synaptobrevin may be critical for its correct localization, as has recently been shown for syntaxin (Ge et al, 2006).…”

Section: Discussionmentioning

confidence: 99%

Determinants of Synaptobrevin Regulation in Membranes

Siddiqui

Vites

Stein³

et al. 2007

MBoC

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Neuronal exocytosis is driven by the formation of SNARE complexes between synaptobrevin 2 on synaptic vesicles and SNAP-25/syntaxin 1 on the plasma membrane. It has remained controversial, however, whether SNAREs are constitutively active or whether they are down-regulated until fusion is triggered. We now show that synaptobrevin in proteoliposomes as well as in purified synaptic vesicles is constitutively active. Potential regulators such as calmodulin or synaptophysin do not affect SNARE activity. Substitution or deletion of residues in the linker connecting the SNARE motif and transmembrane region did not alter the kinetics of SNARE complex assembly or of SNARE-mediated fusion of liposomes. Remarkably, deletion of C-terminal residues of the SNARE motif strongly reduced fusion activity, although the overall stability of the complexes was not affected. We conclude that although complete zippering of the SNARE complex is essential for membrane fusion, the structure of the adjacent linker domain is less critical, suggesting that complete SNARE complex assembly not only connects membranes but also drives fusion. INTRODUCTIONCommunication between neurons is mediated by neurotransmitters that are released from presynaptic nerve endings by Ca 2ϩ -dependent exocytosis of synaptic vesicles. Exocytotic fusion of the vesicle with the synaptic plasma membrane is mediated by the proteins synaptobrevin 2/VAMP2, SNAP-25, and syntaxin 1 (Jahn and Scheller, 2006;Rizo et al., 2006). These proteins are members of the SNARE protein family that are involved in all fusion events of the secretory pathway. SNAREs are characterized by stretches of 60-70 amino acids arranged in heptad repeats, termed SNARE motifs (Weimbs et al., 1997;Fasshauer et al., 1998b;Bock et al., 2001;Day et al., 2006). Syntaxin and synaptobrevin each contain a single SNARE motif that is located adjacent to a C-terminal transmembrane domain. In contrast, SNAP-25 contains two SNARE motifs connected by a palmitoylated linker region that serves as membrane anchor.Synaptobrevin resides in synaptic vesicles, whereas SNAP-25 and syntaxin 1 reside in the plasma membrane. The SNARE motifs of syntaxin, SNAP-25, and synaptobrevin readily assemble into quarternary bundles of ␣-helices (Fasshauer et al., 1997;Sutton et al., 1998). Assembly would thus lead to a tight connection between the membranes. According to this view, assembly is nucleated at the Nterminal ends of the SNARE-motifs and proceeds toward the C-terminal membrane anchor ("zippering"), resulting in a strained "trans"-complex . During membrane merger, the trans-complex would relax into a "cis"-complex in which the transmembrane domains are aligned in parallel. To regenerate the SNAREs for another round of fusion, SNARE complexes need to be disassembled by the AAAϩ-ATPase NEM-sensitive factor (NSF) in conjunction with cofactors termed soluble NSF attachment proteins (SNAPs; Sollner et al., 1993).Although the "zippering" hypothesis of SNARE function has received a lot of experimental support, it is still unclear...

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Section: Discussionmentioning

confidence: 99%

Determinants of Synaptobrevin Regulation in Membranes

Siddiqui

Vites

Stein³

et al. 2007

MBoC

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“…Syntaxin and VAMP proteins are unusual in that, unlike most transmembrane proteins, they lack a signal sequence required for cotranslational insertion into the ER and instead associate with membranes posttranslationally (Kutay et al, 1995). Unlike any other known syntaxin or VAMP protein, syntaxin 17 requires two adjacent hydrophobic domains near its carboxyl terminus for proper membrane anchorage.…”

Section: Discussionmentioning

confidence: 99%

Syntaxin 17 Is Abundant in Steroidogenic Cells and Implicated in Smooth Endoplasmic Reticulum Membrane Dynamics

Steegmaier

Oorschot

Klumperman

et al. 2000

MBoC

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The endoplasmic reticulum (ER) consists of subcompartments that have distinct protein constituents, morphological appearances, and functions. To understand the mechanisms that regulate the intricate and dynamic organization of the endoplasmic reticulum, it is important to identify and characterize the molecular machinery involved in the assembly and maintenance of the different subcompartments. Here we report that syntaxin 17 is abundantly expressed in steroidogenic cell types and specifically localizes to smooth membranes of the ER. By immunoprecipitation analyses, syntaxin 17 exists in complexes with a syntaxin regulatory protein, rsly1, and/or two intermediate compartment SNARE proteins, rsec22b and rbet1. Furthermore, we found that syntaxin 17 is anchored to the smooth endoplasmic reticulum through an unusual mechanism, requiring two adjacent hydrophobic domains near its carboxyl terminus. Converging lines of evidence indicate that syntaxin 17 functions in a vesicle-trafficking step to the smooth-surfaced tubular ER membranes that are abundant in steroidogenic cells.

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“…To assay for membrane anchoring, we performed assays basically as described by Kutay et al (1995), with the following modifications. Translation reactions were treated with 0.2 M Na 2 CO 3 , pH 11.5, for 30 min on ice.…”

Section: Translation/translocation Assays In Vitromentioning

confidence: 99%

A Defective Signal Peptide Tethers the floury-2 Zein to the Endoplasmic Reticulum Membrane

et al. 1997

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The maize (Zea mays L.) floury-2 (f/Z) mutation is associated with a general decrease in storage protein synthesis, altered protein body morphology, and the synthesis of a novel 24-kD a-zein storage protein. Unlike storage proteins in normal kernels and the majority of storage proteins in f/Z kernels, the 24-kD a-zein contains a signal peptide that would normally be removed during protein synthesis and processing. The expected processing site of this a-zein reveals a putative mutation alanine-valine (Ala-Val) that is not found at other junctions between signal sequences and mature proteins. To investigate the impact of such a mutation on signal peptide cleavage, we have assayed the 24-kD f/Z a-zein in a co-translational processing system i n vitro. Translation of RNA from f/Z kernels or synthetic RNA encoding the f/Z a-zein in the presence of microsomes yielded a 24-kD polypeptide. A normal signal peptide sequence, generated by site-directed mutagenesis, restored the capacity of the RNA to direct synthesis of a properly processed protein i n a cell-free system. Both the f/Z a-zein and the f/Z a-zein (Val-Ala) were translocated into the lumen of the endoplasmic reticulum. The processed f/Z a-zein (ValbAla) was localized in the soluble portion of the microsomes, whereas the f/Z a-zein co-fractionated with the microsomal membranes. By remaining anchored t o protein body membranes during endosperm maturation, the f/Z zein may thus constrain storage protein packing and perturb protein body morphology.The maize (Zea mays L.) floury-2 fl2) mutation produces pleiotropic effects that include a soft, starchy endosperm, a reduction in the amount of endosperm storage protein, and changes in the arrangement of proteins within protein bodies (Nelson et al., 1965;Lending et al., 1988). The changes in protein packaging are accompanied by an accumulation of aberrant protein bodies near the nuclei of endosperm cells. Coincident with the appearance of the aberrant protein bodies is an increase in the accumulation

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Transport route for synaptobrevin via a novel pathway of insertion into the endoplasmic reticulum membrane.

Cited by 280 publications

References 44 publications

Determinants of Synaptobrevin Regulation in Membranes

Determinants of Synaptobrevin Regulation in Membranes

Syntaxin 17 Is Abundant in Steroidogenic Cells and Implicated in Smooth Endoplasmic Reticulum Membrane Dynamics

A Defective Signal Peptide Tethers the floury-2 Zein to the Endoplasmic Reticulum Membrane

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