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Positive and negative regulation of a SNARE protein by control of intracellular localization.

ABSTRACT: In Saccharomyces cerevisiae, the developmentally regulated Soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein Spo20p mediates the fusion of vesicles with the prospore membrane, which is required for the formation of spores. Spo20p is subject to both positive and negative regulation by separate sequences in its aminoterminal domain. We report that the positive activity is conferred by a short, amphipathic helix that is sufficient to confer plasma membrane or prospore membrane localization to green fluorescent protein. In vitro, this helix binds to acidic phospholipids, and mutations that reduce or eliminate phospholipid binding in vitro inactivate Spo20p in vivo. Genetic manipulation of phospholipid pools indicates that the likely in vivo ligand of this domain is phosphatidic acid. The inhibitory activity is a nuclear targeting signal, which confers nuclear localization in vegetative cells and in cells entering meiosis. However, as cells initiate spore formation, fusions containing the inhibitory domain exit the nucleus and localize to the nascent prospore membrane. Thus, the SNARE Spo20p is both positively and negatively regulated by control of its intracellular localization.

SUBMITTER: Nakanishi H

PROVIDER: S-EPMC379277 | biostudies-literature | 2004 Apr

REPOSITORIES: biostudies-literature

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Positive and negative regulation of a SNARE protein by control of intracellular localization.

Nakanishi Hideki H de los Santos Pablo P Neiman Aaron M AM

Molecular biology of the cell 20040123 4

In Saccharomyces cerevisiae, the developmentally regulated Soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein Spo20p mediates the fusion of vesicles with the prospore membrane, which is required for the formation of spores. Spo20p is subject to both positive and negative regulation by separate sequences in its aminoterminal domain. We report that the positive activity is conferred by a short, amphipathic helix that is sufficient to confer plasma membrane or pro ...[more]

PMID: 14742704

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Project description:Appropriate protein subcellular localization is essential for proper cellular function. Central to the regulation of protein localization are protein targeting motifs, stretches of amino acids serving as guides for protein entry in a specific cellular compartment. While the use of protein targeting motifs is modulated in a post-translational manner, mainly by protein conformational changes and post-translational modifications, the presence of these motifs in proteins can also be regulated in a pre-translational manner. Here, we investigate the extent of pre-translational regulation of the main signals controlling nucleo-cytoplasmic traffic: the nuclear localization signal (NLS) and the nuclear export signal (NES).Motif databases and manual curation of the literature allowed the identification of 175 experimentally validated NLSs and 120 experimentally validated NESs in human. Following mapping onto annotated transcripts, these motifs were found to be modular, most (73 % for NLS and 88 % for NES) being encoded entirely in only one exon. The presence of a majority of these motifs is regulated in an alternative manner at the transcript level (61 % for NLS and 72 % for NES) while the remaining motifs are present in all coding isoforms of their encoding gene. NLSs and NESs are pre-translationally regulated using four main mechanisms: alternative transcription/translation initiation, alternative translation termination, alternative splicing of the exon encoding the motif and frameshift, the first two being by far the most prevalent mechanisms. Quantitative analysis of the presence of these motifs using RNA-seq data indicates that inclusion of these motifs can be regulated in a tissue-specific and a combinatorial manner, can be altered in disease states in a directed way and that alternative inclusion of these motifs is often used by proteins with diverse interactors and roles in diverse pathways, such as kinases.The pre-translational regulation of the inclusion of protein targeting motifs is a prominent and tightly-regulated mechanism that adds another layer in the control of protein subcellular localization.

Project description:Calcium-triggered exocytotic release of neurotransmitters and hormones from neurons and neuroendocrine cells underlies neuronal communication, motor activity and endocrine functions. The core of the neuronal exocytotic machinery is composed of soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs). Formation of complexes between vesicle-attached v- and plasma-membrane anchored t-SNAREs in a highly regulated fashion brings the membranes into close apposition. Small, soluble proteins called Complexins (Cpx) and calcium-sensing Synaptotagmins cooperate to block fusion at low resting calcium concentrations, but trigger release upon calcium increase. A growing body of evidence suggests that the transmembrane domains (TMDs) of SNARE proteins play important roles in regulating the processes of fusion and release, but the mechanisms involved are only starting to be uncovered. Here we review recent evidence that SNARE TMDs exert influence by regulating the dynamics of the fusion pore, the initial aqueous connection between the vesicular lumen and the extracellular space. Even after the fusion pore is established, hormone release by neuroendocrine cells is tightly controlled, and the same may be true of neurotransmitter release by neurons. The dynamics of the fusion pore can regulate the kinetics of cargo release and the net amount released, and can determine the mode of vesicle recycling. Manipulations of SNARE TMDs were found to affect fusion pore properties profoundly, both during exocytosis and in biochemical reconstitutions. To explain these effects, TMD flexibility, and interactions among TMDs or between TMDs and lipids have been invoked. Exocytosis has provided the best setting in which to unravel the underlying mechanisms, being unique among membrane fusion reactions in that single fusion pores can be probed using high-resolution methods. An important role will likely be played by methods that can probe single fusion pores in a biochemically defined setting which have recently become available. Finally, computer simulations are valuable mechanistic tools because they have the power to access small length scales and very short times that are experimentally inaccessible.

Dataset Information

Positive and negative regulation of a SNARE protein by control of intracellular localization.

Publications

Positive and negative regulation of a SNARE protein by control of intracellular localization.

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