【图文】医学细胞生物学 第06章
基质与内膜_百度文库
两大类热门资源免费畅读
续费一年阅读会员，立省24元！
评价文档：
医学细胞生物学 第06章
基质与内膜
上传于||文档简介
&&医​学​细​胞​生​物​学​教​学​用​P​P​T
大小：34.67MB
登录百度文库，专享文档复制特权，财富值每天免费拿！
你可能喜欢The effect of Sec1p binding was directly tested in an in vitro fusion assay. Fusion is moderately stimulated when recombinant Sec1p is added to a conventional in vitro fusion reaction containing the plasma membrane SNAREs Sso1p, Sec9c, and Snc1p (Fig. 5). However, when Sec1p was prebound in detergent before reconstitution, Sec1p stimulates fusion 2–3-fold compared with t-SNARE complexes that lack bound Sec1p (Fig. 6). Stimulation of fusion by Sec1p is concentration dependent and completely inhibited by soluble Snc2p. These results demonstrate that the presence of Sec1p significantly enhances SNARE-mediated membrane fusion in vitro and is the first in vitro evidence to show a functional consequence of SM protein binding.Sec1p modestly stimulates fusion when added directly to an in vitro fusion assay. Given that Sec1p binds to t-SNARE complexes in detergent (Figs. 3 and 4), we determined if similar Sec1p containing complexes could be reconstituted into liposomes. His6-Sec1p was mixed with His8-Sso1p or His8-Sso1p/GST-Sec9c t-SNARE complexes in the presence of 0.6% octyl-glucoside for ~15 h at 4°C. The overall amount of t-SNARE complex protein added to the reconstitution was reduced to favor the ratio of Sec1p to t-SNARE complex. The detergent solutions were then used to resuspend a lipid film to form unlabeled t-SNARE proteoliposomes. Vesicles were isolated by flotation in a density gradient and analyzed for the presence of specifically bound Sec1p by SDS-PAGE and Coomassie blue staining (Fig. 6 A). We found that significant amounts of His6-Sec1p were isolated with liposomes containing t-SNARE complexes (Fig. 6 A, lanes 2–5); whereas little or no Sec1p was isolated with liposomes containing free His8-Sso1p (Fig. 6 A, lane 1) or protein free liposomes (not depicted).A twofold dilution series of His6-Sec1p protein (700, 350, 175, and 87.5 pmoles) in 400 μl total volume or buffer A200 (400 μl) was mixed with His8-Sso1p (40 μl, 4,715 pmoles) and GST-Sec9c (60 μl, 7,663 pmoles) to form t-SNARE complex in the presence of 0.6% OG. Additionally, Sec1p (700 pmoles) was mixed with Sso1 His8-Sso1p (40 μL, 4,715 pmoles) and buffer A200 (60 μl). Samples were mixed for 16 h at 4°C and reconstituted into unlabeled lipid as previously described (Scott et al., 2003). Sec1p binding was analyzed by SDS-PAGE (Fig. 6 A). Fusion between Snc1p labeled liposomes and t-SNARE unlabeled liposomes containing Sec1p from 0–700 pmoles was monitored as previously described (Weber et al., 1998; Scott et al., 2003). Soluble Snc2p (2 μl, ~660 pmoles) was added to t-SNARE liposomes containing the highest amount of Sec1p for 15 min before adding the labeled Snc1p liposomes to inhibit stimulation of fusion and determine the background level of fusion. The maximal stimulation of fusion by Sec1p was achieved by adding ~700 pmoles of His6-Sec1p protein with t-SNARE proteins before reconstitution was tested with four independent preparations of His6-Sec1p protein. The average fold stimulation with SEM was calculated from these experiments (Fig. 5 C).We next determined the effect of bound Sec1p on SNARE-mediated fusion. Fig. 6 B shows a kinetic fusion reaction with the His8-Sso1p/GST-Sec9c t-SNARE complex with Sec1p bound or without Sec1p (buffer control). The highest concentration of bound Sec1p (Fig. 6 B, closed circles) stimulated fusion roughly threefold over the buffer control (Fig. 6 B, open circles). Soluble Snc2p inhibited fusion in all cases (Fig. 6 B, solid and dashed line) confirming that the Sec1p mediated stimulation is SNARE dependent. Stimulation by Sec1p was examined for four independent preparations of recombinant Sec1p, with an average stimulation by Sec1p of 2.7-fold (Fig. 6 C).Sec1p binding to the t-SNARE complex (mode 2) stimulates fusion, providing a positive regulatory function (Figs. 3 and 6). The binding of other SM proteins to t-SNARE complexes has not been addressed. SM protein binding to the fully assembled SNARE complex (mode 3) occurs for Sec1p (Fig. 3) and Sly1 (Peng and Gallwitz, 2002), although the functional consequences for this interaction mode are not clear. This binding mode may favor trans-SNARE complex assembly (Kosodo et al., 2002), or be involved in SNARE recycling (Carr et al., 1999; Kosodo et al., 2003).SNARE-bound Sec1p strongly stimulates in vitro fusion. A twofold dilution series of Sec1p was bound to t-SNARE complexes (Sso1p/Sec9c) in detergent solution before vesicle reconstitution. (A) Coomassie blue–stained gel of liposomes containing Sec1p bound t-SNARE complexes. Acceptor t-SNARE liposomes containing various amounts of bound His6-Sec1p were resolved on a 10% Bis-Tris NuPAGE gel (Invitrogen) and stained with Coomassie blue. Lane 1 contains 15 μl of liposomes derived from a reaction containing His8-Sso1p (~160 μg, ~4.7 nmol) and 60 μg (700 pmol) of His6-Sec1p. Lanes 2–6 contains 10 μl of liposomes derived from reactions including His8-Sso1p (~160 μg, ~4.7 nmol), GST-Sec9c (~430 μg, ~7.7 nmol) and decreasing amounts of Sec1p: lane 2, ~60, μg, ~700 lane 3, ~30 μg, ~350 lane 4, ~15 μg, ~175 and lane 5, ~7.5 μg, ~88 pmol. Lane 6 contained no Sec1p. Lane 7 contains 0.6 μg (~7 pmol) of recombinant His6-Sec1p. (B) Kinetic fusion graph of Sec1p stimulated fusion. Vesicles (45 μl) containing t-SNARE complexes without Sec1p (open circles, ~13 μg, 145 pmol Sso1p/Sec9c and 42 nmol lipid) and t-SNARE vesicles containing the highest amount of bound Sec1p (closed circles) were mixed with fluorescently labeled vesicles containing the v-SNARE Snc1p (5 μl, ~8.3 μg, 630 pmol Snc1p and 1.95 nmol lipid) and incubated for 120 min at 37°C in a standard fusion reaction. The extent of fusion is represented as rounds of fusion, measured as fold lipid dilution in the reaction. The background values (solid and dashed lines) represent an inhibited reaction containing the same components in addition to the soluble domain of Snc2p to inhibit vesicle fusion. The amount of fusion at 120 min was 1.32 rounds of fusion for the Sec1p stimulated curve (closed circles), 0.42 rounds of fusion for basal fusion (buffer, open circles), and the inhibited fusion background, 0.1 rounds of fusion. Sec1p stimulated fusion ~3.8-fold in this experiment after background subtraction. (C) Average fold stimulation caused by Sec1p. The amount of stimulation by Sec1p was examined using four independent preparations of recombinant His6-Sec1p. This histogram shows that His6-Sec1p stimulates fusion by 2.7-fold on average. The mean ± SEM are represented after the subtraction of an average background of 0.105 rounds of fusion. (D) Sec1p titration showing stimulation is concentration dependent. The extent of fusion observed at 120 min and the amount of Sec1p binding detected to t-SNARE liposomes is represented for independent in vitro fusion experiments relative to the amount of Sec1p added to the reaction. Rounds of fusion at 120 min are shown on the left y axis (open circles) and the amount of Sec1p binding (relative to His8-Sso1p) is quantified on the right y axis (closed circles). Both values are plotted relative to the concentration of Sec1p added to the reaction (nM). The binding values for Sec1p were determined by quantifying the gel shown in Fig. 6 A.The amount of Sec1p bound relative to Sso1p is quantified in Fig. 6 D. At the highest concentration of added Sec1p (Fig. 6 A, lane 2, ~1.4 μM), the Sec1p band is roughly 40% of the Sso1p band. This corresponds to ~16% of the Sso1p containing bound Sec1p when differences in molecular weight are taken into consideration. Although Sec1p binding is substoichiometric, fusion stimulation is concentration dependent. When the levels of Sec1p are increased in the binding reaction, more Sec1p is seen associating with the SNARE liposomes and a proportional increase in fusion is also observed (Fig. 6 D). These data strongly argue that Sec1p directly stimulates SNARE-mediated membrane fusion in vitro.Join ResearchGate to access over 30 million figures and 100+ million publications – all in one place.Copy referenceCopy captionEmbed figurePublished in
Full-text available &
Article & Nov 2004
& The Journal of Cell Biology
+2 more authors...
&...action with their cognate SNARE Syntaxin (Sx), in distinct vesicle transport pathways [3,1415. Deletion and over-expression of SM proteins have shown both positive and negative effects in eac...&Sec/Munc18 (SM) and SNARE proteins are essential for vesicle exocytosis in eukaryotes1234567. The assembly of the fusogenic SNARE complex is regulated in part by SM proteins through interaction with their cognate SNARE Syntaxin (Sx), in distinct vesicle transport pathways [3,1415. Deletion and over-expression of SM proteins have shown both positive and negative effects in each step of vesicle fusion [3,161718.
ABSTRACT: 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.
Full-text available &
Article & Dec 2013
& PLoS ONE
+4 more authors...
0Comments 1Citation
&... as shown by others for the reconstitution of alternative SNARE-dependent lipid-mixing schemes [19,78,79]. Indeed, some bacterial proteins from intracellular pathogens have already been shown to inhibit...&Using this assay, synthetic proteoliposomes with a lipid composition mirroring that of the yeast vacuole and harboring the four full-length, transmembrane domain-containing vacuolar SNARE proteins involved in homotypic fusion, have been shown to rapidly mix lipid bilayers, but only in the presence of the SNARE chaperones Sec17/18p, regulatory lipids such as PI(3)P, and the HOPS complex, thereby recapitulating much of the known biochemical regulation of homotypic vacuole fusion [58,76]. It is also known that proper SNARE complex remodeling and trans-SNARE complex formation must precede fusion in this assay [77], and as shown by others for the reconstitution of alternative SNARE-dependent lipid-mixing schemes [19,78,79]. Indeed, some bacterial proteins from intracellular pathogens have already been shown to inhibit the rate of membrane fusion in these synthetic SNARE-dependent lipid-mixing assays, thus highlighting the ability of some bacteria to directly modulate the core membrane fusion machinery in vitro [62].
ABSTRACT: During infection, the intracellular pathogenic bacterium Legionella pneumophila causes an extensive remodeling of host membrane trafficking pathways, both in the construction of a replication-competent vacuole comprised of ER-derived vesicles and plasma membrane components, and in the inhibition of normal phagosome:endosome/lysosome fusion pathways. Here, we identify the LegC3 secreted effector protein from L. pneumophila as able to inhibit a SNARE- and Rab GTPase-dependent membrane fusion pathway in vitro, the homotypic fusion of yeast vacuoles (lysosomes). This vacuole fusion inhibition appeared to be specific, as similar secreted coiled-coiled domain containing proteins from L. pneumophila, LegC7/YlfA and LegC2/YlfB, did not inhibit vacuole fusion. The LegC3-mediated fusion inhibition was reversible by a yeast cytosolic extract, as well as by a purified soluble SNARE, Vam7p. LegC3 blocked the formation of trans-SNARE complexes during vacuole fusion, although we did not detect a direct interaction of LegC3 with the vacuolar SNARE protein complexes required for fusion. Additionally, LegC3 was incapable of inhibiting a defined synthetic model of vacuolar SNARE-driven membrane fusion, further suggesting that LegC3 does not directly inhibit the activity of vacuolar SNAREs, HOPS complex, or Sec17p/18p during membrane fusion. LegC3 is likely utilized by Legionella to modulate eukaryotic membrane fusion events during pathogenesis.
Full-text available &
Article & Feb 2013
& PLoS ONE
+2 more authors...
0Comments 11Citations
&...malian tissues. This is consistent with the known regulation of Munc18 proteins by phosphorylation [3]. Western blots of C57BL/6 mouse tissues and of the human A549 lung adenocarcinoma cell line were pr...&Note that recombinant Munc18b runs lower (67 kDa) than Munc18b in lung (72–75 kDa), and that the latter runs as a doublet, suggesting that Munc18b undergoes multiple post-translational modifications in mammalian tissues. This is consistent with the known regulation of Munc18 proteins by phosphorylation [3]. Western blots of C57BL/6 mouse tissues and of the human A549 lung adenocarcinoma cell line were probed with M18-A antibodies (A) or M18b-O antibodies (B) as described in the main text.
File available &
Data & Jun 2012
+14 more authors...
0Comments 0Citations
oror log in with【图文】医学细胞生物学 第06章
基质与内膜_百度文库
两大类热门资源免费畅读
续费一年阅读会员，立省24元！
评价文档：
医学细胞生物学 第06章
基质与内膜
上传于||文档简介
&&医​学​细​胞​生​物​学​教​学​用​P​P​T
大小：34.67MB
登录百度文库，专享文档复制特权，财富值每天免费拿！
你可能喜欢