Crystallization and preliminary X-ray diffraction of the Munc18c–syntaxin4<sub>1–29</sub>complex

Latham, Catherine F.; Hu, Shu Hong; Gee, Christine L.; Armishaw, Christopher J.; Alewood, Paul F.; James, David E.; Martin, Jennifer L.

doi:10.1107/s1744309107022361

Cited by 5 publications

(4 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The recombinant Munc18c proteins purified from bacterial expression cultures (HLMunc18c and HMunc18c) can be crystallized in the presence of the Sx4 N-peptide (not shown), under conditions used to crystallize Munc18c derived from baculovirus expression [45] , [48] . Finally, we showed using a pulldown assay that Munc18c (de-tagged) produced from bacterial expression interacts with pre-assembled SNARE complex ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Milligram Quantities of Homogeneous Recombinant Full-Length Mouse Munc18c from Escherichia coli Cultures

et al. 2013

Self Cite

View full text Add to dashboard Cite

Vesicle fusion is an indispensable cellular process required for eukaryotic cargo delivery. The Sec/Munc18 protein Munc18c is essential for insulin-regulated trafficking of glucose transporter4 (GLUT4) vesicles to the cell surface in muscle and adipose tissue. Previously, our biophysical and structural studies have used Munc18c expressed in SF9 insect cells. However to maximize efficiency, minimize cost and negate any possible effects of post-translational modifications of Munc18c, we investigated the use of Escherichia coli as an expression host for Munc18c. We were encouraged by previous reports describing Munc18c production in E. coli cultures for use in in vitro fusion assay, pulldown assays and immunoprecipitations. Our approach differs from the previously reported method in that it uses a codon-optimized gene, lower temperature expression and autoinduction media. Three N-terminal His-tagged constructs were engineered, two with a tobacco etch virus (TEV) or thrombin protease cleavage site to enable removal of the fusion tag. The optimized protocol generated 1–2 mg of purified Munc18c per L of culture at much reduced cost compared to Munc18c generated using insect cell culture. The purified recombinant Munc18c protein expressed in bacteria was monodisperse, monomeric, and functional. In summary, we developed methods that decrease the cost and time required to generate functional Munc18c compared with previous insect cell protocols, and which generates sufficient purified protein for structural and biophysical studies.

show abstract

Section: Resultsmentioning

confidence: 99%

Milligram Quantities of Homogeneous Recombinant Full-Length Mouse Munc18c from Escherichia coli Cultures

et al. 2013

Self Cite

View full text Add to dashboard Cite

show abstract

“…SM and SNARE protein interactions are specific, such that Munc18a and Munc18b only bind to syntaxins 1 and 3 whereas Munc18c only binds to syntaxins 2 and 4 [35] . The N-terminal 29 residues of syntaxin 4 have been shown to be required for binding to Munc18c and the three-dimensional structure of the complex has been elucidated [26] , [36] , [37] . Since we found that efficient surface delivery and basolateral sorting of syntaxin 4 depend on its N-terminal region, and our results confirm that syntaxin 4-Δ29 is not able to bind to Munc18c, this may suggest that formation of the syntaxin 4/Munc18c complex is necessary for surface delivery and/or basolateral sorting of syntaxin 4.…”

Section: Discussionmentioning

confidence: 99%

Basolateral Sorting of Syntaxin 4 Is Dependent on Its N-terminal Domain and the AP1B Clathrin Adaptor, and Required for the Epithelial Cell Polarity

et al. 2011

View full text Add to dashboard Cite

Generation of epithelial cell polarity requires mechanisms to sort plasma membrane proteins to the apical and basolateral domains. Sorting involves incorporation into specific vesicular carriers and subsequent fusion to the correct target membranes mediated by specific SNARE proteins. In polarized epithelial cells, the SNARE protein syntaxin 4 localizes exclusively to the basolateral plasma membrane and plays an important role in basolateral trafficking pathways. However, the mechanism of basolateral targeting of syntaxin 4 itself has remained poorly understood. Here we show that newly synthesized syntaxin 4 is directly targeted to the basolateral plasma membrane in polarized Madin-Darby canine kidney (MDCK) cells. Basolateral targeting depends on a signal that is centered around residues 24–29 in the N-terminal domain of syntaxin 4. Furthermore, basolateral targeting of syntaxin 4 is dependent on the epithelial cell-specific clathrin adaptor AP1B. Disruption of the basolateral targeting signal of syntaxin 4 leads to non-polarized delivery to both the apical and basolateral surface, as well as partial intercellular retention in the trans-Golgi network. Importantly, disruption of the basolateral targeting signal of syntaxin 4 leads to the inability of MDCK cells to establish a polarized morphology which suggests that restriction of syntaxin 4 to the basolateral domain is required for epithelial cell polarity.

show abstract

“…Met-1 was included in the N-peptide for direct comparison with similar studies using N-peptides but may not be present in mature Stx4. The process of producing diffraction-quality crystals is reported in detail elsewhere (38). The crystals used to measure the diffraction data reported here were prepared from purified Munc18c (14 mg/ml in 25 mM Hepes, pH 7.0/150 mM NaCl/2 mM 2-mercaptoethanol) mixed with a 10-fold molar excess of the N-peptide.…”

Section: Methodsmentioning

confidence: 99%

Structure of the Munc18c/Syntaxin4 N-peptide complex defines universal features of the N-peptide binding mode of Sec1/Munc18 proteins

Latham

Gee

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

130

148

View full text Add to dashboard Cite

Sec1/Munc18 proteins (SM proteins) bind to soluble NSF attachment protein receptors (SNAREs) and play an essential role in membrane fusion. Divergent modes of regulation have been proposed for different SM proteins indicating that they can either promote or inhibit SNARE assembly. This is in part because of discrete modes of binding that have been described for various SM/SNARE complexes. One mode suggests that SM proteins bind only to Syntaxins (Stx) preventing SNARE assembly, whereas in another they facilitate SNARE assembly and bind to SNARE complexes. The mammalian cell surface SM protein Munc18c binds to an N-peptide in Stx4, and this is compatible with its interaction with SNARE complexes. Here we describe the crystal structure of Munc18c in complex with the Stx4 N-peptide. This structure shows remarkable similarity with a yeast complex indicating that the mode of binding, which can accommodate SNARE complexes, is highly conserved throughout evolution. Modeling reveals the presence of the N-peptide binding mode in most but not all yeast and mammalian SM/Stx pairs, suggesting that it has coevolved to fulfill a specific regulatory function. It is unlikely that the N-peptide interaction alone accounts for the specificity in SM/SNARE binding, implicating other contact surfaces in this function. Together with other data, our results support a sequential two-state model for SM/SNARE binding involving an initial interaction via the Stx N-peptide, which somehow facilitates a second, more comprehensive interaction comprising other contact surfaces in both proteins. crystallography ͉ protein:protein interactions ͉ vesicle trafficking I ntracellular trafficking relies on membrane fusion, a tightly regulated process that requires the formation of a coiled coil complex between helical motifs originating from soluble NSF attachment protein receptor (SNARE) proteins on vesicle and target membranes (1-3). Sec1/Munc18 proteins (SM proteins) play a fundamental role in this process by interacting with SNARE protein(s) and regulating the formation of SNARE complexes (4, 5). SM proteins have been identified in organisms from yeast to humans, and loss-of-function mutants lead to severe impairment in vesicle fusion (6).Despite their obvious importance, the nature of SNARE regulation by SM proteins remains controversial, in that both positive and negative regulatory roles have been reported (6, 7). Possibly the most compelling evidence in support of negative regulation is the observation that the mammalian Syntaxin1a (Stx1a) SNARE protein involved in synaptic neurotransmission exists in both an ''open'' (SNARE complex-compatible) and a ''closed'' (SNARE complexincompatible) conformation (8, 9). The crystal structure of Stx1a in complex with the SM protein Munc18-1 (Munc18a, nSec1) reveals that Munc18-1 embraces a closed conformation preventing Stx1a from forming the SNARE complex required for vesicle fusion (10). On the other hand, deletion of Munc18-1 results in impaired exocytosis in mouse neurons and chromaffin cells (11, 12),...

show abstract

Crystallization and preliminary X-ray diffraction of the Munc18c–syntaxin4_1–29complex

Cited by 5 publications

References 11 publications

Milligram Quantities of Homogeneous Recombinant Full-Length Mouse Munc18c from Escherichia coli Cultures

Milligram Quantities of Homogeneous Recombinant Full-Length Mouse Munc18c from Escherichia coli Cultures

Basolateral Sorting of Syntaxin 4 Is Dependent on Its N-terminal Domain and the AP1B Clathrin Adaptor, and Required for the Epithelial Cell Polarity

Structure of the Munc18c/Syntaxin4 N-peptide complex defines universal features of the N-peptide binding mode of Sec1/Munc18 proteins

Contact Info

Product

Resources

About

Crystallization and preliminary X-ray diffraction of the Munc18c–syntaxin41–29complex

Cited by 5 publications

References 11 publications

Milligram Quantities of Homogeneous Recombinant Full-Length Mouse Munc18c from Escherichia coli Cultures

Milligram Quantities of Homogeneous Recombinant Full-Length Mouse Munc18c from Escherichia coli Cultures

Basolateral Sorting of Syntaxin 4 Is Dependent on Its N-terminal Domain and the AP1B Clathrin Adaptor, and Required for the Epithelial Cell Polarity

Structure of the Munc18c/Syntaxin4 N-peptide complex defines universal features of the N-peptide binding mode of Sec1/Munc18 proteins

Contact Info

Product

Resources

About

Crystallization and preliminary X-ray diffraction of the Munc18c–syntaxin4_1–29complex