2001;61:3443-3449.Cancer Res
Xiao Huan Liang, Jie Yu, Kristy Brown, et al.
Required for Its Autophagy and Tumor Suppressor Function
Beclin 1 Contains a Leucine-rich Nuclear Export Signal That Is
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[CANCER RESEARCH61,3443–3449,April15,2001]
Beclin1Contains a Leucine-rich Nuclear Export Signal That Is Required for Its Autophagy and Tumor Suppressor Function1
Xiao Huan Liang,Jie Yu,Kristy Brown,and Beth Levine2
Departments of Medicine[X.L.,J.Y.,B.L.]and Pathology[K.B.],Columbia University College of Physicians&Surgeons,New York,New York10032 ABSTRACT
Beclin1encodes a Bcl-2-interacting coiled-coil protein with autophagy
and tumor suppressor function and is monoallelically deleted in40–75%
of sporadic human breast and ovarian cancers.Beclin1contains a leucine-
rich nuclear export signal motif raising the possibility that its autophagy
and/or tumor suppressor function may require regulated,CRM1-depen-
dent,nucleocytoplasmic transport.In this study,we show that wild-type
Beclin1colocalizes with both intracytoplasmic organelles and nuclei in
COS7monkey kidney and MCF7human breast carcinoma cells.Inhibi-
tion of CRM1-dependent nuclear export with leptomycin B or mutation of
the nuclear export signal motif of Beclin1results in predominantly
nuclear localization.Unlike wild-type Beclin1,the nuclear export mutant
of Beclin1fails to promote nutrient deprivation-induced autophagy and
fails to inhibit in vitro clonigenicity and in vivo tumorigenicity of MCF7
cells.Thus,beclin1has a leptomycin B-sensitive leucine-rich nuclear
export signal that is required for its autophagy and tumor suppressor
function.These findings suggest that the CRM1nuclear export pathway
may be important in the functional regulation of autophagic growth
control.
INTRODUCTION
Leucine-rich short amino acid sequences responsible for efficient
nuclear export have been identified in a number of different cellular
and viral proteins(reviewed in Refs.1–4).The first identified exam-
ple of a leucine-rich NES3was a minimal11-amino acid peptide
(LQLPPLERLTL)in the HIV-1Rev protein in which mutation of
critical leucines abolished export activity(5,6).Functional NESs that
resemble the Rev NES“consensus”sequence(Lx
(2–3)Lx
(2–3)
LxL)
have since been detected in other viral and cellular proteins required for RNA export[e.g.,human T-cell lymphotropic virus1Rex(7,8), herpes simplex virus ICP27(9),ribosomal protein L5(10)],cellular proteins involved in signal transduction[e.g.,PKI(11),c-ABL(12), I?B?(13),MAPKK(14)],transcription factors[e.g.,TFIIIA(15)], tumor suppressors[e.g.,p53(16)],and oncogenes[e.g.,hdm2(17)]. The Rev-type NES forms a complex with the nuclear export receptor, CRM1,and Ran GTP(18–23).Leptomycin B,a fungicide,inhibits the formation of this complex by targeting CRM1and blocks nuclear export of NES-containing proteins(21,24).
The use of NESs and the CRM1export pathway has been most extensively studied in the context of viral and cellular RNA export (reviewed in Ref.25).However,the increasing number of different types of identified proteins with Rev-type NESs suggests that the CRM1export pathway is important in the regulation of other eukary-otic cellular functions,including kinase regulation,signal transduc-tion,and growth control.For example,PKI,an inhibitor of the protein kinase protein kinase A,binds to the active catalytic[C]subunits of cyclic AMP in the nucleus,leading to the exposure of its nuclear export signal,rapid export of the C subunit-PKI complex,and termi-nation of signaling(1).Similarly,the NES of I?B?is also postulated to play a role in a negative feedback loop,in which it terminates nuclear factor-?B signaling once in the nucleus(1).The NES of the oncogene hdm2is required for its ability to block p53-mediated transcriptional activation and to accelerate p53export and ubiquitin-mediated degradation(17).p53export may also be regulated by its own intrinsic NES,and a model has been proposed in which NES masking by tetramer formation regulates the subcellular localization and activity of the p53tumor suppressor(16).
A novel potential use for the Rev-related nuclear export pathway is suggested by a structural analysis of human Beclin 1.Beclin1 contains a short leucine-rich amino acid sequence that resembles the Rev-type NESs and it is the first identified mammalian protein to mediate autophagy(26),a process of bulk protein degradation that is required for differentiation,survival during nutrient deprivation,and normal growth control(reviewed in Refs.27–29).Fourteen gene products have been identified in yeast that are required for autophagy (30),including a serine/threonine protein kinase(31)and two proteins involved in a ubiquitin-like protein conjugation system(32).How-ever,far less is known about the molecular events controlling auto-phagy in mammalian cells.The presence of a Rev-type NES in a mammalian autophagy protein raises the interesting possibility that the Rev-related nuclear export pathway may be used for autophagy regulation.
Beclin1was originally isolated in a yeast two-hybrid screen for Bcl-2-interacting proteins and it encodes a M
r
60,000predicted coiled-coil protein(33).Human Beclin1shares24.4%amino acid identity(and39.1%conservation)with a yeast gene product,Apg6/ Vps30p,that is required for both nitrogen deprivation-induced auto-phagy(34)and for proper vacuolar protein sorting(35).In gene transfer studies,beclin1promotes autophagy,but not vacuolar protein sorting,in yeast with a targeted disruption of apg6/vps30(26).Fur-thermore,beclin1gene transfer increases basal levels of autophagy and nutrient deprivation-induced autophagy in human breast carci-noma cells that lack detectable levels of endogenous Beclin1protein. The mechanisms by which Beclin1promotes autophagy in yeast or mammalian cells are not yet understood.
The autophagy function of Beclin1may play a role in negative regulation of tumorigenesis.Beclin1maps to a tumor susceptibility locus on chromosome17q21and is frequently monoallelically deleted in human breast,ovarian,and prostate cancers(reviewed in Ref.36), indicating that it may be a tumor suppressor.Although biallelic mutations of beclin1have not as of yet been demonstrated in human cancer,two lines of evidence suggest that functional inactivation of beclin1may contribute to tumorigenesis.First,Beclin1protein is expressed ubiquitously in all normal breast epithelial cells,but fre-quently has low or undetectable expression in malignant breast epi-thelial cells(26).Second,beclin1gene transfer in human breast carcinoma cells results in the loss of malignant morphological prop-erties,decreased rates of cell proliferation,impaired clonigenicity in vitro,and impaired ability to form tumors in nude mice(26).Thus,
Received8/31/00;accepted2/19/01.
The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with 18U.S.C.Section1734solely to indicate this fact.
1Supported by American Cancer Society Grant RPG-98-339-CCG,NIH Grant RO1 CA84254,and a Mallinckrodt Scholar Award(to B.L.).
2To whom requests for reprints should be addressed,at Department of Medicine, Columbia University College of Physicians&Surgeons,P&S8-463,630West168th Street,New York,NY10032.Phone:(212)305-7312;Fax:(212)305-7290;E-mail: levine@https://www.doczj.com/doc/c114446732.html,.
3The abbreviations used are:NES,nuclear export signal;mt NES,mutant nuclear export signal;3-MA,3-methyladenine;EBSS,Earle’s balanced salt solution;ER,endo-plasmic reticulum;Ab,antibody;PKI,protein kinase inhibitor;I?B,inhibitor?B.
Beclin1,an autophagy protein,can also function as a negative regulator of mammary cell growth and tumorigenesis.
Nothing is known about structural domains of Beclin1that are important for autophagy and tumor suppressor function.Aside from limited homology within the central coiled-coil domain with myosin-like proteins,Beclin1shares no significant homology with any mammalian proteins of known function(33).However,amino acids 180–189of human Beclin1contain a core of closely spaced leucine residues that conform to the consensus motif of the Rev family of NESs.Therefore,we evaluated whether Beclin1contains an NES and if so,whether the NES is required for its autophagy and tumor suppressor function.
MATERIALS AND METHODS
Plasmid https://www.doczj.com/doc/c114446732.html,ing the previously described plasmid pSG5/ flag-beclin1(33)as a template,two-step PCR mutagenesis was used to introduce two leucine3alanine substitutions at amino acid positions184and 187of Beclin1to generate mtNES Beclin1.The cDNA encoding mtNES beclin1was cloned into PCR vector(Invitrogen,San Diego,CA),sequenced using the dideoxychain termination method,and subsequently cloned into pSG5(Stratagene,La Jolla,CA)or pTRE(Clontech Laboratories,Palo Alto,CA).
Cell Lines.COS7monkey kidney cells and MCF7human breast carcinoma cells were obtained from American Type Culture Collection(Manassas,VA) and MCF7/tet-off cells were obtained from Clontech Laboratories;all cells were grown according to the suppliers’instructions.
Transfections.For transient expression,the plasmids pSG/flag-beclin1or pSG5/flag-mtNES beclin1were transfected into COS7cells using SuperFect polycationic reagent(Qiagen,Valencia,CA)and the plasmids pTRE/flag-beclin1and pTRE/flag-mtNES beclin1were transfected into MCF7/tet-off cells using PerFect lipid(Invitrogen)according to the manufacturer’s instruc-tions.For stable expression,MCF7/tet-off cells were cotransfected with pTK-Hyg and pTRE/flag-beclin1or pTRE/flag-mtNES beclin1and maintained in 200?g/ml hygromycin and2?g/ml tetracycline.Hygromycin-resistant clones were screened for inducible wild-type flag-Beclin1or flag-mtNES Beclin1 protein expression following tetracycline withdrawal by immunoblot analysis using M2antiflag epitope monoclonal Ab(1:200;Kodak,Rochester,NY)and peroxidase-conjugated horse antimouse IgG(1:2000)with the enhanced chemiluminescent method of detection(Amersham,Arlington Heights,IL).A monoclonal antiactin Ab(1:400;Boehringer Mannheim,Indianapolis,IN)was used as a control for protein loading in immunoblot analysis.
Immunofluorescence Studies.COS7and MCF7cells were grown on glass chamber slides,transfected with plasmids(2?g per4.0-cm2well) expressing wild-type Beclin1or mtNES Beclin1,and fixed24h after transfection with100%ethanol.Alternatively,leptomycin B was added at a concentration of2.5ng/ml(gift from Minoru Yoshida,University of Tokyo, Tokyo,Japan)24h after transfection,and cells were fixed8h after leptomycin B treatment(32h after transfection).To detect flag-Beclin1and flag-mtNES Beclin1,cells were stained with M2antiflag Ab(1:20)and FITC-conjugated horse antimouse Ab(1:40;Vector Laboratories,Burlingame,CA).The nuclei of all cells were labeled with Hoechst33258(1ng/ml).Double immunofluo-rescent staining was also performed using either a polyclonal Ab against peptide SSR?(1:50)(37)to detect the ER or a polyclonal anti-lamin B Ab(38) (1:100)to detect the perinuclear membrane(gifts from Howard Worman, Columbia University,New York,USA)followed by rhodamine-conjugated goat antirabbit Ab.To colabel mitochondia and Beclin1,live transfected COS7and MCF7cells were incubated with a MitoTracker probe(400n M; Molecular Probes,Eugene,OR)for30min and then fixed and stained with M2 antiflag as described above.For dual-labeling analysis,cells were examined using a Zeiss LSM410scanning confocal microscope equipped with an argon-krypton laser.The percentage of cells demonstrating nuclear staining was calculated by analyzing200–300transfected cells per transfection experiment; results represent the mean?SE for six independent transfection experiments. Cells demonstrating FITC immunoreactivity that colocalized with Hoechst 33258were scored as positive.
Autophagy Analysis.Five days following tetracycline removal from the media,MCF7/tet-off clones stably transfected with empty pTRE(MCF7.control cells),pTRE/flag-beclin1(MCF7.beclin1cells),or pTRE/flag-mtNES beclin1 (MCF7.mtNES beclin1cells)were grown for4h in either(a)EBSS?10% serum and complete amino acids,(b)EBSS,or(c)EBSS following a30-min pretreatment with10m M3-MA.Cells were fixed with2.5%glutaraldehyde in 0.1M Sorenson’s buffer(pH7.2)for1h,postfixed with1%OsO
4
in0.1M Sorenson’s buffer,and embedded in Lx-112(Ladd Research Industries,Bur-lington,VT)and Embed-812(EM Science,Fort Washington,PA).Thin sections were cut on MT-7000RMC,stained with uranyl acetate and lead citrate,and examined by transmission electron microscopy using a JEOL JEM-1200EXII.The number of autophagic vacuoles per cell was determined for100cells/clone for each treatment group in a blinded fashion to calculate the mean number of autophagic vacuoles per cell.Autophagic vacuoles were defined as double-membrane vacuolar structures containing recognizable cy-toplasmic contents using a magnification of?50,000.
Clonigenicity and Tumorigenicity Assays.To measure in vitro clonige-nicity,MCF7.control,MCF7.beclin1,and MCF7.mtNES beclin1clones were seeded at a density of5?104cells/well in triplicate35-mm wells in semi-solid media(0.3%agar in DMEM with20%fetal bovine serum).The number of anchorage-independent colonies(with?20cells/colony)per well was counted at21days.
To measure in vivo tumorigenicity,5-week-old female NCR nude mice (Taconic Farms,Germantown,NY)were implanted with1.7mg of estrogen/ 60-day release pellets(Innovative Research of America,Toledo,OH)and injected s.c.with5?106MCF7tumor cells.Mice were monitored for the development of tumors and tumor size was measured in two dimensions [length(a)and width(b)].Tumor volume was calculated according to V?ab2/2.Mice were necropsied after8weeks,and mean tumor volumes were determined for the subgroup of mice with tumors present upon necropsy.All animal studies were performed in accordance with Institutional Animal Care and Use Committee guidelines.
RESULTS
Subcellular Localization of Beclin1.We examined the subcellu-lar distribution of wild-type Beclin1in transfected COS7cells and MCF7cells(Figs.1and2).Since antibodies are not yet available that detect Beclin1by immunofluorescent staining,we used flag epitope-tagged constructs.[In previous studies,flag epitope-tagged Beclin1 has been shown to have autophagy and tumor suppressor function(26) and demonstrates an immunofluorescent staining pattern in trans-fected cells similar to that observed with immunoperoxidase staining of endogenous Beclin1in human brain and breast tissue(26,33)].In flag-beclin1-transfected COS7cells,the majority of cells demon-strated granular cytoplasmic staining suggestive of localization within intracytoplasmic organelles(Fig.2A,top row)and very rare cells (?1%)demonstrated any nuclear staining.Dual-labeling experiments showed that the FITC-staining of flag-Beclin1colocalized with a MitoTracker,a rhodamine dye that targets to the mitochondria,and with antibodies to SSR?(37)and lamin B(38)that label the ER and perinuclear membrane,respectively(Fig.1A).The flag-Beclin1stain-ing was specific in that no immunostaining was observed in COS7 cells transfected with an empty vector and stained with antiflag M2 Ab(data not shown).Thus,Beclin1is infrequently observed in the nucleus of COS7cells and is predominantly associated with intracy-toplasmic structures including mitochondria,ER,and the perinuclear membrane.The association of Beclin1with these organelles is similar to that previously reported for the Beclin1-interacting proteins Bcl-2
and Bcl-x
L
(39,40).
In flag-beclin1-transfected MCF7cells,a higher percentage (46.5?4.2%)of cells demonstrated some nuclear staining(Fig.2B, top row).In cells with exclusive cytoplasmic or nuclear and cytoplas-mic staining,the cytoplasmic distribution of Beclin1was similar to that observed in COS7cells(Fig.1B).Flag-Beclin1colocalized with MitoTracker as well as with antibodies to the ER and perinuclear membrane.Thus,MCF7cells differed from COS7cells with respect
to the amount of Beclin 1nuclear staining but not with respect to the subcellular distribution of cytoplasmic Beclin 1.
Taken together,these results indicate that Beclin 1demonstrates both cytoplasmic and nuclear localization with cell type-specific differences in the frequency of steady-state nuclear localization.Leptomycin B Blocks the Nuclear Export of Beclin 1.Given the localization of Beclin 1in both nuclei as well as intracytoplasmic organelles,coupled with the presence of a leucine-rich NES motif spanning amino acids 180–189,we hypothesized that Beclin 1may undergo CRM1-dependent nuclear export.To examine this hypothe-sis,we evaluated the effects of leptomycin B,an inhibitor of CRM1-dependent nuclear export (21,24),on the subcellular localization of flag-Beclin 1in COS7and MCF7cells (Fig.2,middle row ).In both cell types,treatment with leptomycin B resulted in the nuclear local-ization of Beclin 1in the majority of transfected cells.At 8h after leptomycin B treatment,exclusive cytoplasmic staining was observed in only 4.0?0.7%of leptomycin B-treated COS7cells as compared to 98.3?0.8%of untreated COS7cells,and in 1.5?0.3%of leptomycin B-treated MCF7cells as compared to 53.5?5.9%of untreated MCF7cells.Consistent with previous reports indicating that leptomycin B results in nuclear retention of NES-containing proteins within a very short period of time (5–90min)(21,41–43),we did not observe any significant differences in the percentage of COS7or MCF7cells with nuclear Beclin 1staining at 2,4,6,8,12,and 18h after treatment (data not shown).The increased nuclear localization of Beclin 1in both COS7and MCF7cells following leptomycin B treatment is consistent with a leptomycin B-sensitive,CRM1-depen-dent pathway of nuclear export for Beclin 1.
Mutation of the NES of Beclin 1Blocks Its Nuclear Export.To determine whether the NES motif of human Beclin 1has
nuclear
Fig.1.Beclin 1colocalizes with mitochondria,ER,and perinuclear membrane markers in COS7and MCF7cells.Confocal scanning laser microscopy images of COS7cells (A )and MCF7cells (B )transfected with flag-beclin1.Left column of each panel ,FITC antiflag immunostaining to detect flag-Beclin 1.Middle column of each panel ,staining with rhodamine-labeled markers of cytoplasmic organelles,including MitoTracker to detect mitochondria (top ),anti-SSR ?to detect ER (middle ),and anti-lamin B to detect perinuclear membrane (bottom ).Right column of each panel ,overlay of FITC and rhodamine staining;yellow ,colocalization of flag-Beclin 1and cytoplasmic
organelle.
Fig.2.Leptomycin B treatment and mutation of the Beclin 1NES alters the subcellular localization of Beclin 1in COS7and MCF7cells.COS7cells (A )and MCF7cells (B )transfected with wild-type flag-Beclin 1(top and middle rows of each panel )in the absence (top rows of each panel )or presence of leptomycin B (middle rows of each panel )or transfected with mtNES flag-Beclin 1(lower rows of each panel ).Images shown represent FITC an-tiflag immunostaining to detect flag-Beclin 1(left column of each panel )or Hoechst 33258staining to detect nuclear DNA (right columns of each panel ).
export function,we examined the subcellular localization of an NES mutant of Beclin 1.Fig.3shows an alignment of the NES of human Beclin 1and mouse Beclin 1with other well-characterized leucine-rich NESs.Based upon mutational analyses of NESs in HIV-1Rev and PKI (5,11),we predicted that mutation of the two leucine residues at 184and 187should block nuclear export of Beclin 1.Therefore,we mutated these two leucine residues to alanine resi-dues (mtNES Beclin 1)and examined the subcellular distribution of flag-mtNES Beclin 1(Fig.2,bottom row ).The subcellular local-ization of mtNES flag-Beclin 1contrasted markedly with wild-type flag-Beclin 1in untreated COS7and MCF7cells,but was similar to that observed following leptomycin B treatment of wild-type beclin 1-transfected cells.Nuclear staining of mtNES1was observed in 99.1?0.9%of transfected COS7cells and in 97.2?5.3%of transfected MCF7cells,as compared to nuclear staining of wild-type Beclin 1in ?1%of transfected COS7cells and in 46.5?4.2%of transfected MCF7cells.The nuclear localization of mtNES flag-Beclin 1in almost all transfected cells indicate that the leucines at amino 184and 187in human Beclin 1are critical for its nuclear export.
Mutation of the NES of Beclin 1Blocks Its Autophagy Func-tion.The above studies demonstrate that Beclin 1has a leucine-rich NES that undergoes CRM1-dependent nuclear export.To determine whether the NES of Beclin 1is required for its autophagy function,we compared the ability of wild-type Beclin 1and mtNES Beclin 1to promote nutrient deprivation-induced autophagy in MCF7cells.Se-rum and amino acid deprivation is a potent inducer of autophagy in normal cells (reviewed in Ref.27),but not in MCF7cells that lack detectable endogenous Beclin 1expression (26).However,enforced wild-type Beclin 1expression increases basal levels of autophagy in MCF7cells and promotes nutrient deprivation-induced autophagy that can be inhibited by the nucleotide derivative 3-MA (26).
To permit conditional expression of an antiproliferative gene,we stably transfected MCF7/tetoff cells with a tetracycline-repressible vector,pTRE,containing no insert (referred to as MCF7.control cells),wild-type flag-beclin 1(referred to as MCF7.beclin1cells),or flag-mtNES beclin 1(referred to as MCF7.mtNES beclin1cells).One MCF7.control clone,one MCF7.beclin1clone,and two MCF7.mtNES beclin1clones which expressed different levels of mtNES Beclin 1protein were chosen for further analysis (Fig.4A ).After normalization using an actin control,densimetric analysis revealed comparable levels of mtNES Beclin 1expression in MCF7.mtNES beclin1clone 17cells and wild-type Beclin 1ex-pression in MCF7.beclin1clone 6cells (data not shown).
MCF7clones were either maintained in normal growth conditions or subjected to 4h of serum and amino acid starvation and analyzed by electron microscopy for the presence of autophagic vacuoles (Fig.
4,B and C ;see Fig.4C for representative images of the ultrastructural appearance of MCF7clones in normal growth or starvation condi-tions.)Ultrastructural analysis is the most definitive method to iden-tify autophagic vacuoles,which are defined as double membrane-bound 0.3–2mm vesicles with clearly recognizable cytoplasmic contents (44,45).An example of an autophagic vacuole which would be counted in our quantitative electron microscopy analysis is shown in Fig.4C ,lower right panel .Using quantitative electron microscopy analysis,we found that the baseline numbers of autophagic vacuoles per cell were comparable in the MCF7.control clone and the two MCF7.mtNES beclin1clones,which was significantly less than that observed in the MCF7.beclin1clone (Fig.4B ).Furthermore,serum and amino acid deprivation increased the number of autophagic vacu-oles in MCF7.beclin1cells,but not in MCF7.mtNES beclin1cells or in MCF7.control cells.The nutrient deprivation-induced autophagy in MCF7.beclin1cells was completely blocked by pretreatment with the autophagy inhibitor 3-MA.Thus,mtNES beclin 1,unlike wild-type beclin 1,failed to promote basal or nutrient deprivation-induced autophagy in MCF7cells.
Mutation of the NES of Beclin 1Blocks Its Tumor Suppressor Function.To evaluate the tumor suppressor activity of mtNES beclin 1,we compared MCF7.control,MCF7.beclin1,and MCF7.mtNES beclin1cells with respect to their in vitro clonigenicity and tumorigenicity in nude mice (Fig.5).MCF7.mtNES beclin1cells formed colonies in soft agar as efficiently as MCF7.control cells,whereas MCF.beclin 1cells were severely impaired in anchorage-independent growth (Fig.5A ).The inability of mtNES beclin 1to suppress clonigenicity in soft agar was associated with an inability to suppress MCF7tumorigenicity in nude mice.After an 8-week observation period,only 10%of mice injected with MCF7.beclin1cells developed tumors,as compared with 78%of mice injected with MCF7.control cells or 71–86%of mice injected with MCF7.mtNES beclin1cells (Fig.5B ).In addition,the tumor volume was significantly less in the tumor that did develop in the mouse injected with MCF7.beclin1cells (32mm 3)as compared with the mean tumor volume in mice injected with MCF7.control (218?65mm 3)or MCF7.mtNES beclin1cells (381?170mm 3)for clone 17,758?282mm 3for clone 23;(P ?0.001,t test).These data demonstrate that mtNES beclin 1,unlike wild-type beclin 1,lacks tumor suppressor function in MCF7human breast carcinoma cells.DISCUSSION
Our results demonstrate that Beclin 1has a functionally important leucine-rich NES.Inhibition of the CRM1nuclear export pathway by leptomycin B or mutation of the conserved leucine residues within the Beclin 1NES blocks the nuclear export of Beclin 1.Mutation of the NES interferes with the two known functions of the protein,e.g.,the promotion of nutrient-deprivation induced autophagy and the suppression of mammary cell tumorigenesis.These results suggest a role for the Beclin I NES and the Rev-related nuclear export pathway in the regulation of autophagy and cell growth in mammalian cells.The finding of leucine-rich NESs in signal transduction molecules (such as MAPKK,PKI,and I ?B)suggests that nuclear export signals are important in coordinating the cell’s response to stress and external stimuli.Since autophagy is tightly regulated in response to environ-mental stimuli (e.g.,amino acid availability,hormones,growth fac-tors)(reviewed in Refs.27and 29),our findings lead to the hypothesis that nuclear cytoplasmic shuttling of autophagy proteins may be a cellular mechanism for the control of autophagy.Interestingly,we have noted that the yeast counterpart of Beclin 1,Apg6/Vps30p,has a sequence near its NH 2terminus (LDPSLEGLSL)that conforms to the Rev-type NES consensus sequence (Lx (2–3)Lx (2–3)LxL).Al-though it is not yet known whether this sequence is an NES
for
Fig.3.Alignment of the Beclin 1NES with other known leucine-rich Rev-type NESs.Numbers indicate the position of the amino acids within the proteins.Conserved hydro-phobic residues (primarily leucines)are shown in boldface .Underlined amino acids represent those mutated in human mtNES Beclin 1.In the NES consensus sequence,X is any amino acid and L (leucine)can be substituted by other large hydrophobic residues.
Apg6/Vps30p,the evolutionarily conservation of this motif in the yeast and mammalian counterparts of an autophagy gene suggest that it may play an essential functional role.
Our findings also support the hypothesis that nuclear cytoplasmic shuttling is an important mechanism for regulating cell growth.Pre-vious studies have demonstrated that nuclear cytoplasmic shuttling may be critical for the regulation of p53tumor suppressor function;the CRM1nuclear export pathway is used by cellular [e.g.,hdm2(17),mdm2(46)]and viral oncogenes [e.g.,HPV E6(46)]to target p53for ubiquitin-mediated degradation.Recent evidence also suggests that a hydrophobic NES with loose similarity to the Rev-type NESs may play a role in the cell cycle-dependent nuclear localization of the mitotic cyclin (47–49).Before mitosis,cyclin B is rapidly exported from the nucleus resulting in a steady-state cytoplasmic localization and this export is blocked by leptomycin B.Phosphorylation of cyclin B1at the beginning of mitosis blocks its interaction with CRM1and prevents its return to the cytoplasm (49).The failure of a nuclear export-deficient mutant of Beclin 1to suppress the tumor-forming potential of human breast carcinoma cells further suggests that nuclear cytoplasmic shuttling is critical for the function of cell growth regu-latory proteins.However,in contrast to the p53tumor suppressor which is functionally inactivated by transport to the cytoplasm,nu-clear export of Beclin 1is required for its tumor suppressor activity.This requirement for an intact nuclear export signal of Beclin 1in tumor suppressor function is most likely interrelated with the require-ment for an intact nuclear export signal in autophagy function.Based upon inverse correlations between cellular malignant transformation and autophagic capacity,it has been postulated that autophagy is an important mechanism for negative growth control (reviewed in Ref.28).In addition,inactivation of the autophagy inhibitory TOR/RAFT1signaling pathway results both in autophagy and a G 0arrest (50,51).Our earlier finding that enforced expression of Beclin 1both promotes nutrient deprivation-induced autophagy and inhibits tumorigenicity of human breast carcinoma cells suggested that beclin 1may provide a genetic link between autophagy and tumor suppressor pathways (26).The identification of a common structural requirement in Beclin 1,an intact Rev-type NES,for both its autophagy and tumor suppressor function further suggests that these two functions may be
interrelated.
Fig.4.Mutation of the Beclin 1NES abrogates its autophagy function in MCF7cells.A ,Western blot analysis of flag-Beclin 1and actin expression in different MCF7clones (clone number designated following hyphen).Blots shown represent identical membrane probed with antiflag M2Ab and then reprobed with antiactin Ab (clone number desig-nated following hyphen).B ,quantitative effects of Beclin 1and mtNES Beclin 1on basal and nutrient deprivation-induced autophagy of MCF7cells.Bars ,mean (?SE)number of autophagic vacuoles per cell for cells growing in normal media (gray ),for cells subjected to 4h of serum and amino acid deprivation (black ),and for cells pretreated for 30min with 10m M 3-MA and subjected to 4h of serum and amino acid deprivation in the presence of 3-MA (white ).Mean was determined by count-ing the total number of autophagic vacuoles in each cell for 100cells/clone per treatment.C ,represent-ative electron micrographs of MCF7.control (clone 38;left column )MCF7.mtNES beclin1(clone 17;middle column )or MCF.beclin1(clone 6;right column )grown either in nutrient-rich media (top row )or subjected to 4h of serum and amino acid deprivation (middle row and bottom row ,respec-tively).Bottom row ,higher magnification of boxed area in middle row .?,a representative autophagic vacuole that would be counted in the experiment shown in B .f ,5?m;Ⅺ,0.5?m.
If this proves to be correct,the loss of autophagy function resulting from failure to undergo nuclear export could be directly responsible for the loss of tumor suppressor function of the nuclear export-deficient mutant of Beclin 1.However,we cannot exclude the possi-bility that Beclin 1has two independent functions,both of which require the leucine-rich NES motif.
Although we speculate that the loss of autophagy and tumor sup-pressor function of mtNES Beclin 1results from aberrant nucleocy-toplasmic transport,it is also possible that this domain of Beclin 1functions through independent mechanisms to regulate autophagy and tumor suppressor function.For example,the leucine-rich NES of p53is located within its tetramerization domain,and a model has been proposed in which p53tetramerization occludes its NES and ensures nuclear retention of the DNA-binding form (16).It is possible that the leucine-rich NES of Beclin 1,which is located within the coiled-coil domain of the protein,is important for homodimerization or for heterodimerization with other proteins and that such protein-protein interactions could modulate the function of Beclin 1.The yeast homologue of Beclin 1,Apg6p,has been shown to interact with another coiled-coil protein Apg14p (34).However,to date,there is no known mammalian homologue of Apg14p and the only known Beclin 1-interacting proteins are Bcl-2and Bcl-x L which bind to a region of Beclin 1(amino acids 88–150)that is upstream of the NES motif (33).To further understand the role of the NES of Beclin 1in its autophagy and tumor suppressor functions,it will be necessary to identify the effects of stimuli that promote and inhibit autophagy on the subcellular localization of Beclin 1.The yeast homologue,Apg6/Vps30p,has been previously shown to associate with intracellular membranes (34),and,in this study,we found that Beclin 1associates with intracytoplasmic organelles such as the ER,mitochondria,and perinuclear membrane.Since structural algorithmic analyses of Beclin 1fail to reveal any hydrophobic transmembrane domains (33),the association of Beclin 1with intracytoplasmic membranes presumably occurs through association with other integral membrane proteins.Given that the double membranes of autophagic vacuoles are primar-ily derived from invaginations of the ER membrane (44,45),it is possible that Beclin 1must be present in the ER for autophagy to proceed.According to this model,the nuclear compartmentalization of Beclin 1would represent a mechanism for blocking autophagy from occurring in the cytoplasm.An alternative explanation is that the nuclear export of Beclin 1is required to facilitate the degradation of another autophagy-inhibitory protein;this model would be analogous to the scenario observed with hdm2and p53(17).
Viewed in the context of these models,the cell type-specific dif-
ferences that we observed in the subcellular localization of wild-type Beclin 1may have functional significance for the regulation of auto-phagy and tumorigenesis.MCF7cells are a human breast carcinoma cell line that fails to undergo nutrient deprivation-induced autophagy,and we found that the percentage of cells with nuclear localization of Beclin 1in MCF7cells was significantly higher than in COS7cells.Although COS7cells are transformed by SV40T antigen,we have also observed a predominantly cytoplasmic pattern of staining of flag-Beclin 1in nontransformed cells such as BHK cells (33)and NIH3T3cells.4The molecular basis for the increased nuclear local-ization of Beclin 1in MCF7as compared to other cell types is unknown;it could reflect cell type-specific differences in as of yet undefined modifications of Beclin 1that influence nuclear export (e.g.,binding to other proteins,phosphorylations)or cell type-specific differences in CRM1or other components of the intrinsic nuclear export machinery.It is already known that the beclin 1gene is frequently monoallelically deleted (36)and that Beclin 1protein expression is frequently decreased in human breast cancer (26).It is possible that the blockade of Beclin 1nuclear export represents an additional mechanism to inactivate its tumor suppressor function in human breast carcinoma cells.ACKNOWLEDGMENTS
We thank Howard Worman for helpful discussions and for providing antibodies and Minori Yoshida for providing leptomycin B.
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教学目标： 1.让学生掌握英语学习中的五种基本句型； 2.学会分辨句子属于哪种句型； 3.懂得使用不同的句型造句； 教学重点与难点： 1.五种句型的分析与理解； 2.句型4（主+动+宾+补）与句型5（主+动+宾+宾）的掌握与比较； 教学方法： 1.ppt演示； 课堂练习： 一、下列的句子属于哪种类型 1. The sun rises 2. She is walking along the lake. 3. I like this book very much. 4. That man seems kind 5. He bought his sister a piano. 6. She kept us waiting for over three hours. 7. Let me give you a hand. 8. We tried to make her happy. 二、请说出五个句子，并说出属于哪种句型 家庭作业： 1.每个句型各举出3个句子 2.翻译句子
导入主题：我们现在能用英语写简单的作文，也可以用英语进行简单的交流与沟通，可是大家知道英语中有多少种基本句型吗？那这些句型分别是什么？ （让学生自由思考、讨论，引出今天的课题，英语学习中的五种基本句型）1.Subject (主语) ＋Verb (谓语) 此句型的特点是:谓语动词是不及物动词,本身能表达完整的意思,后面不需跟宾语,但有时可跟副词、介词短语等作状语。如: He laughed. John has read widely. He lives in London. 2. Subject(主语) ＋Verb (谓语) ＋Object (宾语) 此句型的特点是:谓语动词是及物动词,不能表达完整的意思,必须跟有一个宾语。如: Our team beat all the others. 3. Subject (主语) ＋Link. V(系动词) ＋Predicate(表语) 此句型的特点是:谓语动词是连系动词,不能表达完整的意思,必须加上一个表明主语特征、身份、状态的表语。常见的系动词有:be(是),become(成为),get(变得),turn(变得),grow(变得),look(看起来),feel(感到),smell(闻起来),taste(尝起来),sound(听起来),seem(似乎),keep(保持),stay(保持)等。如: The rose smells sweet. 4. Subject(主语)＋Verb (动词)＋Object (宾语)＋Complement(补语) 此句型的特点是:谓语动词虽然跟有一个宾语,但意思还不完整,必须加上另外一个成分(宾语补足语)对宾语进行补充说明。可以用作宾语补足语的有:名词、形容词、不定式、动名词、分词、介词短语等。如: We must keep our school clean. They made him their monitor. 5. Subject(主语)＋Verb(谓语)＋Indirect object(间接宾语)＋Direct object (直接宾语) 此句型的特点是:谓语动词跟有两个宾语,这两个宾语都是动作的对象或承受者,其中指人的是间接宾语,指物的是直接宾语。当间接宾语放在直接宾语之后时,通常需要加介词for或to。可跟双宾语的动词 有:answer,bring,buy,find,get,give,lend,make,pass,pay,send,show,sing,take,teach,tell, write等。如: Mr. Li told us an interesting story. Would you please give this dictionary to Li Hua？ 【注】S=Subject(主语). V=Verb(谓语动词). P=Predicative(表语). O=Object(宾语).
固定句型及固定搭配归纳 一、接不定式（而不接动名词）作宾语的常用动词（意思是此类动词后面要接动词时需用to do 形式，而不能用V.ing形式） afford to do sth. 负担得起做某事 agree to do sth. 同意做某事arrange to do sth.安排做某事ask to do sth. 要求做某事demand to do sth. 要求做某事choose to do sth. 决定做某事decide to do sth. 决定做某事determine to do sth. 决心做某expect to do sth. 期待做某事help to do sth. 帮助做某事wish to do sth. 希望做某事hope to do sth. 希望做某事learn to do sth. 学习做某事manage to do sth. 设法做某事offer to do sth. 主动提出做某plan to do sth. 计划做某事prepare to do sth. 准备做某事pretend to do sth. 假装做某事promise to do sth. 答应做某事refuse to do sth. 拒绝做某事want to do sth. 想要做某事 注：有些不及物动词后习惯上也接不定式，不接动名词： fail to do sth. 未能做某事long to do sth. 渴望做某事happen to do sth. 碰巧做某事hesitate to do sth. 犹豫做某struggle to do sth. 努力做某事 二、接不定式作宾补的常用动词 advise sb. to do sth. 建议某人做某事 allow sb. to do sth. 允许某人做某事 ask sb. to do sth.请（叫）某人做某事 cause sb. to do sth. 导致某人做某事 command sb. to do sth. 命令某人做某事 = order sb. to do sth.
2015年高中英语选修六Unit 4作业题6套（人教版有答案和解释）Unit 4 Global warming Period One Warming Up & Reading Ⅰ.用适当的介、副词填空 1．Thatprobablydoesnotseemmuch________youorme，butitisarapidincreasewhencompared________othernaturalchange s. 2．Sohowhasthiscome________anddoesitmatter? 3．Allscientistssubscribe________theviewthattheincrease____ ____theearth’stemperatureisdue________theburning________fo ssilfuels________coal，naturalgasandoiltoproduceenergy. 4．Theproblembeginswhenweaddhugequantities________extracarb ondioxideintotheatmosphere. 5．Weknowthatthelevels________carbondioxidehaveincreasedgre atlyoverthelast100________150years. 6．Hefoundthatbetweentheseyearsthecarbondioxide________thea tmospherewent________fromaround315parts________around370par tspermillion. 7．Theyalsoagreethatitistheburningofmoreandmorefossilfuelst hathasresulted________thisincreaseincarbondioxide. 8．However， theattitudesofscientists________thisrisearecompletelydiffer ent. 9．________theonehand， Dr.Fosterthinksthatthetrendwhichincreasesthetemperatureby5d egreeswouldbecatastrophe. 10．________theotherhand，therearethose，likeGeorgeHambley， whoareopposed________thisviewandbelievethatweshouldnotworry ________highlevelsofcarbondioxide________theair. Ⅱ.佳句翻 译与仿写1．Thereisnodoubtthattheearthisbecomingwarmerandthatitishum anactivitythathascausedthisglobalwarmingratherthanarandombu tnaturalphenomenon. 翻译：
常用口语： 1. I’m not myself！我烦透了！ 2. Don’t bother me！别烦我！ 3. Give me five more minutes, please. 再给我五分钟时间，好吗？ 4. How did you sleep? 你睡的怎么样？ 5. Don’t hog the bathroom! 别占着卫生间了！ 6. Don’t hog the shower! 别占着浴室了！ 7. Don’t hog my girlfriend! 别缠着我的女朋友了！ 8. Get outta there! 快出来！ 9. I will treat you. 我请客。 10. What are you in the mood for? 你想吃什么？ 11. Who is gonna drive? / Who’s driving? 谁来开车？ 12. You know what I mean? 你明白我的意思吗？ 13. Could you run that by me again?
你能再说一遍吗？ 14. So what you are trying to say is... 那么，你想说的是…… 15. Whadja do last night? 昨晚你干嘛去了？ Whadja=What did you 16. Didja have a good time? 玩的开心吗？ didja=did you 17. Where wouldja like to go tonight? 今晚你想上哪儿？ Wouldja=Would you 18. I am running late. 我要迟到了。 19. I’ve gotta get outta here. 我得离开这儿了。 20. I’ve gotta catch the bus. 我要去赶公共汽车了。 21. gotta=got to wanna=want to gonna=going to 22. Yo—taxi! 嗨，出租车！ 23. Where to ? (你)要去哪儿？
补充：英语写作常用句型： 句子写的好坏，是文章写作成败的关键。以下一些惯用句型，实用性较强。 一。开头句型： 1．As far as…is concerned,…就……而论 When it comes to sth/doing sth. 当谈论到…… 1)As far as the role of information in the future is concerned, I believe that information will play a more and more important part in people’s decision-making. 2)As far as what was mentioned above is concerned, how can we ignore the advantages travelling brings forth? 就前面所提及的而言，我们怎么可以忽视旅游带来的裨益呢？ 2．It goes without saying that…不用说 1)It goes without saying that practice makes perfect. 2)It goes without saying that reading makes a full man. 3. It can be said with certainty that…可以肯定的说 1)It can …… that cultivating a hobby can add fun to our life. 2)It can ….… that because of the knowledge, we can now enjoy a comfortable life which is brought about by advanced technology. 可以肯定地说，正由于知识我们才能享受科技进步所带来的舒适生活。 4. As the proverb says, …有句谚语说 As the saying gose, …俗话说 As the saying puts it, …俗话说 1) As the proverb says, “Honesty is the best policy.”诚实为上策。 2) As………goes, “Diligence makes up for deficiencies.”俗话说：“勤能补拙”。 3)An English proverb says, “Lost time is never found again.” 英国有句谚语说的好：“光阴如流水，一去不复回。” 5. It has to be noticed that …必须注意的是…… 1)It has to be noticed that the air we breathe is already seriously polluted by the exhaust factories and vehicles give off. 必须注意的是：我们呼吸的空气已经被工厂和交通工具所释放的废气严重污染了。 2) It has to be noticed that market all over the country are swamped with fake and poor quality products and this has greatly harmed the state’s interests and people’s health. 必须注意的是：假冒伪劣的产品充斥全国市场，这种情况大大损害了国家利益和人 民的健康。 6. It’s generally recognized that …人们普遍认为 1) It’s generally recognized that college students shouldn’t try to reach after what is beyond their grasp when they choose to find a good job after graduation.人们普遍认为大学生 在大学毕业后想找一个好工作时，不应该追求超出他们能力以外的目标。 2)It’s generally recognized that lazy hands makes a man poor, while diligent hands bring
专业四级辅导：s Test Eight It is the first time +that分句 在“It is/was/will be the first time+that分句”结构中，当主句动词为is/will be 时，that分句动词一律用现在完成体，引导词that可以省略。例如： It is the first time I’ve been here. Don’t forget, it’ll be the first time I’ve spoken in public. 当主句动词为was时，that分句动词通常用过去完成体。例如： It was the first time she’d been at a summer school and she thoroughly enjoyed it. 近义词辨析 fast, hasty, quick, rapid, speedy, swift 这组词均含有“迅速的”，“快捷的”的意思。 fast 强调速度快，且保持不变；又指钟表的时间超过准确的时间。 We took a fast train to Beijing.我们坐快车来北京。 hasty 指匆忙、急促，涉及因此而引起的慌乱、疏漏。 Don’t arrive at hasty conclusions.别匆忙下结论。 quick 强调动作、行为发生得突然且持续时间短。指人时，暗示聪明、领悟快。 The tiger took a quick leap at the sheep.老虎猛地向羊扑去。
rapid 指就整个过程来讲速度快，但并不一定指这一过程的速度始终均匀不变。 The growth of the economy has been rapid in recent years.近年来，经济增长速度很快。 speedy 指人处理问题迅速或物体运转速度快。 His accusations brought a speedy denial.他的指控马上遭到了否认。 swift 与fast和quick同义，但带有文学色彩。 Eagles are swift in flight.鹰飞得很快。 全真模拟试题 1. It was requested that all of the equipment ____in the agreed time. A. Erected B. would be erected C. be erected D. will be erected 2. The man sitting opposite me smiled dreamily, as if ____something pleasant in the past. A. to remember B. remembered C. having been remembered D. remembering 3. I ____him the Christmas gift by mail because he came home during the Christmas
第一节动词的时态 一、一般现在时： 1、由when、as soon as、the minute、the moment、till、until等引起的时间状语从句，以及由if、unless、provided that等引起的条件状语从句常常用一般现在时态表示将来的动作，而主句则用 一般将来时态。 例：They will go home for winter vocation as soon as they finish their exams. 2、当表示普遍的真理或者众所周知的客观事实，常常用一般现在时态。例：The earth is round. 地球是圆的。 二、一般过去时：区分三个短语的用法： 1、used to do sth：过去常常做某事。 2、be/get used to doing sth：习惯做某事。 3、be used to do sth：被用于做某事。 三、一般将来时： 1、be to+动词原形：表示安排或计划好了的动作。例：The Third-Ring Road is to be open to traffic before National Day. 2、be about to+动词原形：表示即将发生的动作。例：The lecture is about to begin.讲座即将 开始。 3、一些表示动作趋势，如开始、终结，以及一些表示动作方向，如往来的动词，常常用现在进行 时态表示按照安排将于将来发生的事情，这类动词常见的有如：start，go，leave，come，arrive等。例：We are leaving for Beijing tomorrow. 我们明天动身去北京。 四、进行时态： 重点区分when和while引起的时间状语的用法。 When表示时间上的点，在考试中其引导的时间状语从句多翻译为“这时？”，主句多用进行时态；while引导的时间状语从句多翻译为“正当……时”，该从句用进行时态。例：One of the guards was sleeping when the general came in, which made him very angry. I fell and hurt myself while I was playing tennis. 五、现在完成时：
中考英语-句子的固定搭配（含答案）-专题练习 一、单选题 1.It's half past seven. It's time to . A. have a breakfast B. have breakfast C. have the breakfast D. has this breakfast 2.I'm looking forward to __________my grandparents this summer holiday. A. see B. sees C. saw D. seeing 3.It takes about 30 minutes _______ a bicycle from my home to school. A. ride B. riding C. to ride D. rides 4.Could you please the dishes? A. to do B. do C. to make D. makes 5.It is very interesting for me ______________ to cook food. A. learned B. learn C. to learn D. learning 6.——I feel a bit hungry now. ——Why not_____ for dinner with us? A. go B. did you go C. to go D. do you go 7.—_______ the weather _______ Hangzhou? —Sunny but cold. A. Where's; of B. What's; in C. How's; in D. How's; of 8.China ________ food and drinks ________ the people in Philippines(菲律宾) last year. A. provided, for B. provides, with C. provided, with D. provides, for 9.It's your turn ________ the classroom. A.clean B.to cleaning C.to clean D.cleans 10.While we down the street, the accident happened. A. walked B. are walking C. were walking 11.She had great difficulty ________ the suitable food on the menu in that restaurant. A. finding B. find C. found D. to find 12.What's with you? A. wrong B. the wrong C. matter D. up 13. When will you return to your hometown? __________. A. Not until the end of this month B. Until next week C. A few days ago D. A few weeks before 14.It usually takes about ten minutes ____to school. A. by bike B. on a bike C. to ride D. ride 15.Xiao Li was supposed __________ her homework before nine o'clock. But she couldn't.
四年级下册英语复习资料 Unit1Myschool(我的学校) 一、单词： 楼层：firstfloor一楼secondfloor二楼 教室：teachers’office教师办公室library图书馆playground操场computerroom 计算机房musicroom音乐教室artroom美术教室gym体育馆 其他：nextto在近旁homework作业class班；班级forty四十way方向 指示代词：this这，这个(通常指空间上离我们较近的单数物品) that那，那个(通常指空间上离我们较远的单数物品) 二、重点句型： 1.询问教室位置：Whereisthelibrary图书馆在哪里？ It’sonthesecondfloor.它在二楼。 2.猜测教室： Isthistheartroom这是美术教室吗？Yes,itis.No,itisn’t. Isthattheteacher’soffice那是…吗Yes,itis.No,itisn’t. Isitacomputer它是计算机吗？Yes,itis.No,itisn’t. Doyouhaveaplayground你们（学校）有操场吗？ Yes,wedo.No,wedon’t. Howmanystudentsarethereinyourclass你班有多少个学生？ Forty-fivestudents.四十五个学生。 礼貌待客“这边请”Thiswayplease. P5：Let’sdo部分： Gotothelibrary.Readabook.去图书馆，读书。
Gototheteacher’soffice.Sayhello.去教师办公室，说您好！Gototheplayground.Playfootball.去操场，踢足球。Gotothegarden.Watertheflowers.去花园，浇花。 三、四会单词和四会句子 P9:Thisisthelibrary.Thatistheplayground. 这是图书馆。那是操场 Unit2Whattimeisit(几点钟了) 一、词汇（另外要根据时间判断相应的活动a.m.上午p.m.下午）名词词组： 一日三餐：breakfast早餐lunch午餐dinner晚餐 课程：musicclass音乐课PEclass体育课Englishclass英语课 动词词组：getup起床gotoschool去上学gohome回家 gotobed上床睡觉 二、重点句型: 是时候该It’stimefor+名词词组.It’stimeforEnglishclass. 干什么了：It’stimeto+动词词组.It’stimetogotoschool. 询问时间： Whattimeisitnow现在几点钟了？ It’s5o’clock.5点了。 P15：Let’sdo部分. Timeforbreakfast.Let’sdrinksomemilk. 到吃早餐的时间了。让我们喝些牛奶吧。 Timeforl unch.Let’shavesomechicken.
五种基本句型 句子就是由主语、谓语动词、表语、宾语、宾语补足语等组成得,依其组合方式可分为五种基本句型,如下表所示： 注意句子成分得表示法Ｓ:Ｓｕbject(主语) V:Verｂ(动词)O: Ｏbjｅct（宾语) P：Ｐrｅdiｃａtive(表语) OＣ: Oｂｊeｃｔplement(宾语补足语) 五种基本句型见下表： 第１种S+V主＋谓 第2种S+V+Ｏ主+谓+宾 第３种Ｓ+V＋P 主+谓+表 第 4 种S+V+o(间接宾语)＋Ｏ（直接宾语） 第 5 种S+Ｖ＋O＋OC 主+谓＋宾+宾补 第 1 种句型:主语+不及物动词:S+Ｖ?Ｂirｄs fly、鸟飞、--－-- －-主语谓语（不及物动词) Ｈｅrｕns in ｔｈe park、她在公园里跑、----－- -－－－---－--－--主语谓语地点状语(不及物动词)? 此句型就是主语＋不及物动词构成句子得主体部分。不及物动词,后面当然不能直接带宾语了，要补上相应得介词,但就是可以有状语来修饰。上例中得iｎｔｈe ｐark,就是地点状语。 Ｃlass begins、(bｅgin 在句中就是不及物动词) 开始上课。 比较ｗe ｂegiｎOｕｒcｌaｓs at eｉｇht、我们八点钟开始上课。该句属于第 2 种句型,bｅgｉn 在句中就是及物动词，由此可见有些动词既可作及物动词也可以作不及物动词。 第 2 种句型:主语＋及物动词+宾语:S+V＋O My fatｈｅｒreaｄtｈｅｂooｋ、 我父亲读过那本书、?(及物动词)?注意有些不及物动词后面加上介词就可把它瞧成一个及物动词，后面就可以加宾语了。Ｙou mｕst ｌｉｓten ｔｏmｅ、您必须听我得。(Liｓtｅn 就是不及物动词。但加上ｔo 之后,Ｌistｅn ｔｏ可以瞧成一个及物动词) 后面直接带宾语得动词就是及物动词，名词与名词得相当语(如代词、不定式、动名词……)等都可充当宾语。Ｓｈeｌikｅs Engｌisｈ、（名词作宾语） I knｏｗhim vｅrｙwell、(代词作宾语) (同第一种句型一样,本句型可以有状语、定语修饰）Thｅｙwant ｔo gｏ、(不定式作宾语） Hｅstopｐeｄwｒiting、(动名词作宾语) ?第3 种句型:主语＋系动词+表语:S+Ｖ+P Ｈe ｂｅcａme a ｓｃienｔisｔ、她成为一个科学家了?谓语(系动词) be 动词与ｂee 就是英语中常见得系动词,后面必须接表语,才能用来说明主语,表示“…… 就是……”,“……变成……”等意思。 表语通常就是名词或形容词等。Tｈｅy are honｅst、她们就是诚实得。 Hｅｂecameａsｃiｅntisｔ、她成为了一个科学家。 His face goes reｄ、她得脸变红了。Ｉt grew dark、天变黑了。 注意在英语中,除了bｅ动词与bee 属于系动词外，还有一些实义动词,当表示状态存在或表示状态变化时也可以作系动词。这些词有:keeｐ、lｏok 、
英语最基础的语法句型有哪些 英语语法是针对英语语言进行研究后，英语语法系统地总结归纳出来的 一系列语言规则。英语语法的精髓在于掌握语言的使用。 1 英语五种基本句型结构一、句型1：Subject (主语) ＋Verb (谓语) 这种句型中的动词大多是不及物动词，所谓不及物动词，就是这种动词后 不可以直接接宾语。常见的动词如：work, sing, swim, fish, jump, arrive, come, die, disappear, cry, happen 等。如： 1) Li Ming works very hard.李明学习很努力。 2) The accident happened yesterday afternoon.事故是昨天下午发生的。 3）Spring is coming. 4) We have lived in the city for ten years. 二、句型2：Subject (主语) ＋Link. V(系动词) ＋Predicate(表语) 这种句型主要用来表示主语的特点、身份等。其系动词一般可分为下列两 类： (1)表示状态。这样的词有：be, look, seem, smell, taste, sound, keep 等。如： 1) This kind of food tastes delicious.这种食物吃起来很可口。 2) He looked worried just now.刚才他看上去有些焦急。 (2)表示变化。这类系动词有：become, turn, get, grow, go 等。如： 1) Spring comes. It is getting warmer and warmer.春天到了，天气变得越来越暖和。 2) The tree has grown much taller than before.这棵树比以前长得高多了。
2011三星笔试固定搭配必背句型 固定搭配必背句型 1. allow sb to do sth 允许某人去做某事（后接动词不定式） My father allowed me to go out for a walk after finishing my homework. 2. asked sb (not) to do sth 叫某人做事某事（叫某人不要去做某事） My father asked me to study hard. He asked me not to swim alone. be asked to do sth 被叫去做某事/被邀请去做某事 I was asked to have a dinner with them yesterday. 3. be afraid to do sth 害怕做某事 She is afraid to ask me questions. 4. be afaid of doing sth 害怕做某事 I am afraid of going out at night. 5. be afaid of sth 害怕某物 He is afraid of snakes. 6. be amazed to do sth 对做某事感到惊讶 He was amazed to meet the girl there. be amazed at sth 对某事感到惊讶 they were amazed at the news. 7. be busy doing/with sth 忙于做某事（常考） I was busy washing my car at that time. 那时候我正忙于清洗我的车子。 I am busy with my work. 8. be coming/going/leaving/fiying/moving/dying（某些位移动词用进行时态时表将来） the bus is coming/the dog is dying. 9. be excited to do sth 对做……感到兴奋 Jacky was excited to travel there by plane. be excited at sth Lily was excited at his words. be excited about doing sth he was excited about passing the exam without going overing books. 10. be frightened to do sth 害怕去做某事 Sam is frightened to ride a horse. 11. be glad/happy to do sth 高兴去做某事 she is happy to clean the blackboard with me. be pleased to do sth高兴做某事 she was pleased to help the old man yesterday. be pleased with sth 对某事感到高兴/满意 the teacher was pleased with my answer. 12. be interested in sth/doing sth 对某事感兴趣/对做某事感兴趣 she is interested in swimming in the river. My btother is interested in Chinese. 13. be/get ready for/to do sth Be ready for sth 为某事做好了准备 We are ready for the exam.
五种基本句型 句子由主语和谓语两大部分组成。主语结构比较单一，谓语结构则不然，不同类别的谓语动词导致不同的谓语结构，从而形成了不同的句型（Sentence Pattern）。换句话说，不同的句型是由不同类别的谓语动词所决定的，因此，句型又被称为动词句型（Verb Pattern）。语法家们对句型的分类不尽相同，一般认为，现代英语的基本句型主要有五种： 1．“主----系-----表”（SLP）句型： 在这种句型中，谓语动词是以be为主的连系动词（Linking Verb），后面跟主语补语（Subject Complement），如： These oranges have kept fresh. 这些桔子一直很新鲜。 Mary is a student / here /in the room. 玛丽是个学生/在这儿/在房间里。 有的语法家把be后面的副词（短语）或介词短语视为状语，从而形成了“主----动-----状”（SVA）这一在五种基本句型之外的句型（见上面最后一个例句）。 2．“主----动”（SV）句型： 在这种句型中，谓语动词为不及物动词（Intransitive Verb），谓语部分通常只包括限定动词，即使有状语，也属任意性状语，即：去掉后并不影响句子结构和意义的完整，如：This bread won’t keep (for a long time). 这种面包不好（长期）存放。 Tom has left. 前面说过,有些句子中的状语是不可缺少的，这种状语被称为必具性状语，从而构成“主 ----动----状”(SVA)句型，但这仅限于少数几个动词的某些用法，因而不是一种主要句型，如： They are staying in a hotel. 他们暂住在一家旅馆里。 3．“主----动-----宾”（SVO）句型： 在这种句型中，谓语动词为只带一个宾语的及物动词，这种动词被称为单宾语及物动词（Monotransitive Verb），如： He kept the money. 他保存着这些钱。 They have left the hotel. 他们已经离开了那家旅馆。 在这种句型中，状语通常也不是必不可少的，但有少数几个及物动词的某些用法要求必具性状语，从而形成了“主----动-----宾-----状”（SVOA）这一在五种基本句型之外的另一种句型，如： The train leaves London at nine. 这列火车九点钟驶离伦敦。
I It is high time that you went to bed. 该是你睡觉的时候了。 https://www.doczj.com/doc/c114446732.html, Itishightime they arrived. 他们到得正是时候。 https://www.doczj.com/doc/c114446732.html, Itishightime to be setting our faces towards reconstructing our country. 现在正是我们着手重建家园的时候了。 https://www.doczj.com/doc/c114446732.html, Itishightime that we implemented the reform. 是实施改革的时候了。 https://www.doczj.com/doc/c114446732.html, Itishightime that we had our lunch. 该是我们吃午饭的时候了。 https://www.doczj.com/doc/c114446732.html, Itishightime that such practices were ended. 该是结束这种做法的时候了。 https://www.doczj.com/doc/c114446732.html, Itishightime that we began the meeting. 正是我们开会的时间了。 119.75.215.166 Itishightime to begin our plan. 该是开始我们计划的时候了。 https://www.doczj.com/doc/c114446732.html, Itishightime that all the chinese people realized the harm of piracy. 是到了全中国人意识到盗版危害的时候。
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