核糖体 自噬

Both the autophagy and proteasomal pathways facilitate the Ubp3p-dependent depletion of a subset of translation and RNA turnover factors during nitrogen starvation

in Saccharomyces cerevisiae

SHANE P.KELLY1and DAVID M.BEDWELL1,2

1Department of Cell,Developmental and Integrative Biology,2Department of Microbiology,University of Alabama at Birmingham, Birmingham,Alabama35294,USA

ABSTRACT

Protein turnover is an important regulatory mechanism that facilitates cellular adaptation to changing environmental conditions. Previous studies have shown that ribosome abundance is reduced during nitrogen starvation by a selective autophagy mechanism termed ribophagy,which is dependent upon the deubiquitinase Ubp3p.In this study,we asked whether the abundance of various translation and RNA turnover factors are reduced following the onset of nitrogen starvation in Saccharomyces cerevisiae.We found distinct differences in the abundance of the proteins tested following nitrogen starvation:(1)The level of some did not change;(2)others were reduced with kinetics similar to ribophagy,and(3)a few proteins were rapidly depleted.Furthermore, different pathways differentially degraded the various proteins upon nitrogen starvation.The translation factors eRF3and eIF4GI,and the decapping enhancer Pat1p,required an intact autophagy pathway for their depletion.In contrast,the deadenylase subunit Pop2p and the decapping enzyme Dcp2p were rapidly depleted by a proteasome-dependent mechanism. The proteasome-dependent depletion of Dcp2p and Pop2p was also induced by rapamycin,suggesting that the TOR1pathway influences this pathway.Like ribophagy,depletion of eIF4GI,eRF3,Dcp2p,and Pop2p was dependent upon Ubp3p to varying extents.Together,our results suggest that the autophagy and proteasomal pathways degrade distinct translation and RNA turnover factors in a Ubp3p-dependent manner during nitrogen starvation.While ribophagy is thought to mediate the reutilization of scarce resources during nutrient limitation,our results suggest that the selective degradation of specific proteins could also facilitate a broader reprogramming of the post-transcriptional control of gene expression.

Keywords:autophagy;proteasome;nitrogen starvation;translation factors;RNA turnover factors

INTRODUCTION

Macroautophagy(hereafter referred to as autophagy)is an important mechanism used by eukaryotic cells to degrade cy-tosolic contents and recycle the resulting building blocks for the synthesis of new macromolecules during stress con-ditions.Autophagy in yeast occurs mainly in response to nu-trient limitation(Takeshige et al.1992).In this process, portions of the subcellular environment are sequestered into de novo formed double membrane vesicles and routed to the yeast vacuole(or mammalian lysosome)where the contents are degraded.The resulting degradative products are transported back into the cytoplasm through vacuolar permeases to facilitate their reuse in biosynthetic pathways. In this way,autophagy maintains cytoplasmic amino acid lev-els and basal protein synthesis during starvation conditions (Onodera and Ohsumi2005;Yang et al.2006;Yang and Klionsky2007).

The target of rapamycin(TOR1)kinase functions as an important sensor of nitrogen and amino acid availability in eukaryotic cells.Addition of the TOR1inhibitor,rapamycin, induces autophagy during nutrient-rich growth(Noda and Ohsumi1998).TOR1regulates the autophagy pathway by di-rect phosphorylation of Atg13p(Kamada et al.2010).TOR1 kinase activity is rapidly inhibited under starvation condi-tions,allowing dephosphorylated Atg13p to accumulate and induction of autophagy to occur.However,TOR1func-tion is gradually reactivated in an autophagy-dependent manner during prolonged starvation in both yeast and mam-malian cells,suggesting that TOR1reactivation may play a

Corresponding author:dbedwell@https://www.wendangku.net/doc/8510042541.html,

Article published online ahead of print.Article and publication date are at https://www.wendangku.net/doc/8510042541.html,/cgi/doi/10.1261/rna.045211.114.?2015Kelly and Bedwell This article is distributed exclusively by the RNA Society for the first12months after the full-issue publication date(see http:// https://www.wendangku.net/doc/8510042541.html,/site/misc/terms.xhtml).After12months,it is available under a Creative Commons License(Attribution-NonCommercial4.0Inter-national),as described at https://www.wendangku.net/doc/8510042541.html,/licenses/by-nc/4.0/.

898RNA21:898–910;Published by Cold Spring Harbor Laboratory Press for the RNA Society

role in the attenuation of autophagy(Yu et al.2010;Shin and Huh2011).

Until recently,autophagy was thought to facilitate random degradation of cytoplasmic macromolecules and organelles. However,the involvement of ubiquitin as a specificity factor for selective autophagy is emerging,and recent evidence sug-gests that crosstalk exists between proteasome-mediated deg-radation and selective autophagy(Kraft et al.2010).Many examples of selective autophagy have been reported.One of these,ribophagy,specifically degrades ribosomes upon nitro-gen starvation.Both40S and60S ribosomal subunits are de-graded by ribophagy,but only60S ribosomal subunit decay requires the activity of the deubiquitinase Ubp3p(Kraft et al.2008).Kraft et al.(2008)found that Rpl25p and other 60S ribosomal subunit proteins were enriched with ubiquitin conjugates in a ubp3Δstrain during nitrogen starvation,sug-gesting that deubiquitination of ribosomal proteins may facilitate degradation of60S subunits during nitrogen starvation.In addition,Ubp3p has been shown to physically interact with Cdc48p,a key component of the ubiquitin–proteasome system.Cdc48p binds to multiple cofactors,in-cluding the deubiquitinase Ubp3p and the ubiquitin ligase Ufd3p(Rumpf and Jentsch2006;Dargemont and Ossareh-Nazari2012).The interaction of these distinct classes of ubiq-uitin-processing enzymes with Cdc48p is thought to make it a“decision platform”for substrate ubiquitination.Finally, the ribosome-associated E3ubiquitin ligase Ltn1p/Rkr1p was recently found to antagonize Ubp3p activity and act as an inhibitor of ribophagy during growth in nutrient-rich conditions(Ossareh-Nazari et al.2014).

Besides ribosomes,a small subset of cytoplasmic proteins, including eIF4GI,have been shown to undergo selective deg-radation during nitrogen starvation in Saccharomyces cerevi-siae(Berset et al.1998;Gelperin et al.2002;Onodera and Ohsumi2004;Shimobayashi et al.2010).eIF4GI is also de-graded upon rapamycin addition,while other translation ini-tiation factors such as eIF4E and eIF4A remain stable under these conditions in both mammalian and yeast cells(Berset et al.1998;Powers and Walter1999;Kuruvilla et al.2001; Ramirez-Valle et al.2008).The selective degradation of eIF4GI during nitrogen starvation is interesting from the gene regulation standpoint since depletion of eIF4GI in mammalian cells by shRNA knockdown not only decreases the translation of mRNAs involved in cell proliferation,but also promotes the induction of autophagy(Ramirez-Valle et al.2008).Deletion of the yeast TIF4631gene encoding eIF4GI impairs global translation initiation rates and cell growth(Clarkson et al.2010).These results suggest that the abundance of eIF4GI broadly influences the translation of many classes of mRNAs.

Given the previous findings that ribosomes and eIF4GI undergo selective degradation during nitrogen starvation, we examined the fate of14translation and mRNA decay fac-tors under these conditions.We found that two translation factors,eRF3and eIF4GI,are rapidly degraded by autophagy in a manner that requires the ribophagy deubiquitinase Ubp3p.Furthermore,two mRNA turnover factors,Dcp2p and Pop2p,were depleted during nitrogen starvation at a faster rate than eRF3or eIF4GI.The decreased abundance of Dcp2p and Pop2p was Ubp3p-dependent,but also re-quired the proteasome pathway in a manner that was re-pressed by TOR1during nutrient-rich conditions.We also found that Ubp3p itself is depleted during nitrogen starva-tion.Taken together,our data show that Ubp3p mediates the depletion of a subset of translation and RNA turnover factors by both the proteasome and autophagy pathways dur-ing nitrogen starvation.

RESULTS

A subset of translation and RNA turnover factors

are degraded by autophagy during nitrogen starvation

Previous studies have shown that nitrogen starvation causes the selective depletion of ribosomes through a process called ribophagy(Kraft et al.2008;Ossareh-Nazari et al.2010).To initially explore ribophagy in our strains,we monitored the abundance of the endogenous large ribosomal subunit pro-tein Rpl3p,as well as the18S and25S rRNAs during nitrogen starvation.We found that the initial abundance of Rpl3p and both rRNAs was reduced by twofold within24h of the onset of nitrogen starvation(Fig.1A);no further decrease was ob-served after48h of starvation.Thus,ribophagy decreased ri-bosome content by up to twofold in our strains following the onset of nitrogen starvation.Thereafter,ribosome abundance reequilibrated at a new steady-state level.

This decrease in ribosome content upon nitrogen starva-tion led us to inquire about the fate of other proteins associ-ated with the post-transcriptional control of gene expression. To do this,we examined the fate of nine translation factors and five RNA decay factors following the onset of nitrogen starvation.The choice of these proteins was primarily based on the availability of specific antibodies or epitope-tagged ex-pression clones of each.Changes in the abundance of these proteins were compared with the ribophagy control protein Rpl3p,whose abundance routinely decreased10%–25%in the first6h following nitrogen depletion.The cytosolic pro-tein Pgk1p and the mitochondrial protein Tom70p were used as controls and found to be stable under the conditions used (Fig.1B–D).

We found that the abundance of three translation factors (eIF2α,eIF5B,and eEF3)and two RNA turnover factors (Lsm1p and Tpa1p)did not change appreciably during the first6h of nitrogen starvation(Fig.1B).Five other proteins (the translation factors eIF5A,eEF1A,eEF2,eRF1,and the poly(A)binding protein Pab1p)exhibited modest(20%–40%)decreases in abundance following the onset of nitrogen starvation,roughly similar to the ribophagy marker Rpl3p (Fig.1C).To examine the role of autophagy in reducing Selective protein depletion during nitrogen starvation

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the abundance of these latter proteins upon nitrogen starva-tion,we utilized an atg7Δstrain.Atg7p shares homology with E1activating enzymes and mediates the conjugation of Atg12p with Atg5p,and Atg8p with phosphatidylethanol-amine.The absence of Atg7p blocks autophagosome forma-tion,an essential early step of autophagy (Feng et al.2014).We found that the decrease in the abundance of these pro-teins (as well as the appearance of a degradation product for eIF5A)was blocked in the atg7Δstrain in a manner sim-ilar to Rpl3p (Fig.1C).These results suggest that the reduced abundance of these proteins is due to degradation by the autophagy pathway.

Interestingly,we found that the abundance of three other proteins,the translation factors eIF4GI and eRF3,as well as the decapping enhancer Pat1p,was reduced much more rap-idly than Rpl3p,with decreases of four-to fivefold within 4h of nitrogen starvation (Fig.1D,E).The decreased abundance of these proteins was also mediated primarily by autophagy,since they were stabilized in an atg7Δstrain under the same conditions.However,the inability of the atg7Δmutation to completely block the decrease of these proteins may suggest that another mechanism,such as reduced synthesis,may also contribute to their decrease.The degradation of these proteins is specific for nitrogen starvation,since the onset of glucose starvation did not induce eIF4GI degradation as previously reported (Gelperin et al.2002)or eRF3degrada-tion (Fig.1D).These results demonstrate that the abundance of a subset of translation and mRNA turnover factors is rap-idly reduced following the onset of nitrogen starvation,and much of this decrease is dependent upon the autophagy pathway.

The cytoplasm to vacuole (Cvt)pathway is a specialized form of autophagy that delivers at least two hydrolases,α-mannosidase (Ams1)and aminopeptidase I (Ape1),to the vacuole.During assembly of the Cvt complex,Atg19p serves as a cargo receptor (Lynch-Day and Klionsky 2010).To test whether the Cvt pathway also participates in the pref-erential degradation of eIF4GI and eRF3during nitrogen starvation,we examined the effect of an atg19Δmutation on their depletion.We found that the decreased abundance of both proteins was unaffected by the atg19Δmutation (Fig.1F),indicating that the Cvt pathway is not responsible for their turnover.

To confirm that the reduced abundance of eRF3and eIF4GI was mediated (at least in part)by protein degradation,we carried out metabolic pulse-chase experiments using an-tibodies to these proteins (Fig.2).Two identical cultures were labeled for 15min with [35S]-methionine in nitrogen rich conditions.A chase period was then initiated by the addition of excess unlabeled methionine and cells from both cultures were rapidly harvested.Subsequently,one culture was resus-pended in nitrogen rich medium,while the other was shifted to nitrogen-free medium.Aliquots of each were harvested at time points thereafter to quantitate the amount of [35S]-labeled eRF3and eIF4GI remaining.We found that the half-life of both proteins decreased significantly following the onset of nitrogen starvation (Fig.2A,B).These results confirm that an increase in the rate of degradation by auto-phagy plays a significant role in the reduced abundance of eRF3and eIF4GI following the onset of nitrogen

starvation.

FIGURE 1.A subset of translation and RNA turnover factors are rap-idly degraded by autophagy following the onset of nitrogen starvation.(A )Western blots of Rpl3p and Pgk1p,and an RNA gel of rRNA from cells harvested at various times following the onset of nitrogen starvation.Quantitation of Rpl3p (center )and rRNA (right )abundance is also shown.(B )Western blots of translation and RNA turnover factors from a wild-type strain whose abundance did not decrease following a shift to nitrogen starvation (?N).(C )Western blots of translation and RNA turnover factors in wild-type and atg7Δstrains that showed a modest decrease in abundance following a shift to nitrogen starvation (?N).(D )Western blots of translation and RNA turnover factors in wild-type and atg7Δstrains that showed a significant decrease in abun-dance following a shift to nitrogen starvation (?N).A shift to glucose starvation (?Glu)for the indicated times was also examined.(E )Quantitation of Western blots from D for proteins showing the largest changes in abundance after exposure to nitrogen starvation (?N)for the indicated times (compared with the ribophagy control,Rpl3p).(F )Western blots of eRF3and eIF4GI in an atg19Δstrain after exposure to nitrogen starvation (?N)for the indicated times.Quantitation is shown to the right .Protein abundance was normalized to Pgk1p from the same extract as an internal control.All experiments were carried out two or more times with similar results.Bar graphs are plotted as mean ±standard deviation.

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The abundance of Dcp2p and Pop2p is rapidly reduced in a proteasome-dependent manner following the onset of nitrogen starvation

We also examined the abundance of Dcp2p and Pop2p fol-lowing the onset of nitrogen starvation.Dcp2p is the catalytic subunit of the Dcp2p/Dcp1p decapping complex,while Pop2p (also known as Caf1p)is a component of the Ccr4p –Pop2p deadenylase complex.We found that the steady-state level of both proteins was reduced in a rapid manner following the onset of nitrogen starvation (Fig.3A,B),with their levels decreasing by four-to fivefold within 2h.Intriguingly,their degradation was not blocked in the atg7Δstrain,indicating that their depletion was not due to degradation by autophagy.Since autophagy did not mediate the decreased abundance of Dcp2p and Pop2p,we next asked if the proteasome was re-sponsible.We found that addition of the proteasome inhib-itor MG132to the culture medium partially stabilized both proteins during nitrogen starvation (Fig.3C,D).These results suggest that both Dcp2p and Pop2p become substrates for proteasomal degradation following the onset of nitrogen star-vation.To confirm the role of the proteasome,we examined the levels of these proteins in a pre1-1/pre2-2strain,which carries mutations in two of the three catalytic subunits of

the 20S proteasome (Heinemeyer et al.1991,1993).We found that both Dcp2p and Pop2p were partially stabilized in a pre1-1/pre2-2strain when grown at 30°C (a semi-restric-tive temperature)(Fig.3E,F).These results confirm that the rapid decrease in abundance of Dcp2p and Pop2p following the onset of nitrogen starvation is at least partially mediated by the proteasome.

To confirm that the Dcp2p and Pop2p proteins are subject to direct proteasomal degradation under these conditions,

we

FIGURE 2.The stabilities of eRF3and eIF4GI are decreased following the onset of nitrogen starvation.Pulse-chase measurements of protein half-life were carried out for (A )eRF3and (B )eIF4GI.Measurements were carried out twice for each protein and condition with similar

results.

FIGURE 3.Dcp2p and Pop2p are rapidly degraded by the proteasome following the onset of nitrogen starvation.(A )Western blots of Dcp2p and Pop2p abundance in wild-type and atg7Δstrains harvested at the in-dicated times following the onset of nitrogen starvation (?N).(B )Quantitation of Western blots from panel A .(C )Western blots and quantitation of Dcp2p in a wild-type strain harvested at various times following a shift to nitrogen starvation (?N).(D )Western blots and quantitation of Pop2p in a wild-type strain harvested at various times following a shift to nitrogen starvation (?N).In panels C and D ,MG132(100μM)was added at the onset of nitrogen starvation as indi-cated.(E )Western blots and quantitation of Dcp2p in wild-type and pre1-1,pre2-2strains harvested at various times following a shift to ni-trogen starvation (?N).(F )Western blots and quantitation of Pop2p in wild-type and pre1-1,pre2-2strains harvested at various times following a shift to nitrogen starvation (?N).The experiments shown in panels E and F were carried out at 30°C.For Dcp2p blots,the arrow indicates the Dcp2p band,while the small star indicates a nonspecific band recog-nized by the HA antibody.For quantitation,protein abundance was normalized to Pgk1p from the same extract as an internal control.All experiments were carried out two or more times with similar results.Bar graphs are plotted as mean ±standard deviation.

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again attempted pulse-chase experiments as described above. However,we were unable to detect either of these proteins by immunoprecipitation.Therefore,while direct proteasomal degradation of Dcp2p and Pop2p is the most direct interpre-tation of these results,we cannot yet exclude an indirect role of the proteasome in their decreased steady-state abundance following the onset of nitrogen starvation.

Ubp3p is required for selective degradation

of translation and RNA turnover factors by

the autophagy and proteasomal pathways

during nitrogen starvation

Activation of the ribophagy pathway following the onset of nitrogen starvation requires the deubiquitinase Ubp3p and its associated cofactor,Bre5p(Cohen et al.2003;Kraft et al. 2010).Since our results showed that eRF3,eIF4GI,and Pat1p are rapidly degraded by autophagy following the onset of nitrogen starvation,we next asked whether their turnover was also Ubp3p-dependent.We found that the rapid decrease in both eRF3and eIF4GI abundance was moderated in both ubp3Δand bre5Δstrains(Fig.4A,B),suggesting that deubi-quitinase activity of the Ubp3p–Bre5p complex facilitates degradation of these proteins.In contrast,the ubp3Δmuta-tion did not alter the degradation of Pat1p.We also examined whether Ubp3p is required for the proteasomal degradation of Dcp2p and Pop2p during nitrogen starvation.We found that both proteins were strongly stabilized in the ubp3Δstrain (Fig.4A,C).These results indicate that Ubp3p participates in the rapid and selective reduction of a subset of translation and RNA turnover factors by both the autophagy and the proteasomal pathways during nitrogen starvation. Proteins depleted by the proteasome during nitrogen starvation are also reduced following rapamycin addition

Rapamycin is a macrolide antibiotic that mediates rapid TOR1inhibition,which results in the induction of autophagy (Noda and Ohsumi1998).Since our results indicated that Dcp2p and Pop2p are rapidly decreased in a proteasome-dependent manner following the onset of nitrogen starva-tion,we next asked whether rapamycin also induced a similar response.We found that the abundance of Dcp2p and Pop2p also rapidly decreased upon rapamycin treatment(Fig.5A, B).This turnover was blocked by MG132,indicating that their depletion under these conditions was again dependent upon proteasome function.To confirm that TOR1inhibition was required for this decrease,we transformed strains with a plasmid expressing the rapamycin resistant tor1-1allele.The tor1-1mutation prevents TOR1binding by the FKBP-rapa-mycin complex(Schmelzle and Hall2000).Thus,expression of this mutant allele should prevent degradation of Dcp2p and Pop2p upon rapamycin addition if TOR1regulates this pro-teasomal process.Indeed,we found that degradation of both Dcp2p and Pop2p was blocked in strains expressing the tor1-1allele(Fig.5C,D).These results indicate that TOR1activity represses the proteasomal depletion of Dcp2p and Pop2p during growth in nutrient-rich conditions,while TOR1inactivation following nitrogen starvation or rapamy-cin exposure induces selective,rapid depletion of these pro-teins by the proteasome.We note that the smaller decrease in Pop2p abundance observed following rapamycin addition as compared with nitrogen starvation could be due to the fact that rapamycin is a relatively specific inhibitor of the TOR1 pathway,while nitrogen starvation perturbs not only the TOR1pathway,but also other physiological processes.

Ubp3p is depleted during nitrogen starvation and has a genetic interaction with the proteasome

We next examined the abundance of Ubp3p during nitrogen starvation.We found that the level of Ubp3p is

rapidly FIGURE4.The ribophagy factor Ubp3p is required for selective pro-tein degradation by both the autophagy and proteasomal pathways dur-ing nitrogen starvation.(A)Western blots of eRF3,eIF4GI,Pat1p, Dcp2p,and Pop2p abundance in wild-type,ubp3Δ,and bre5Δstrains harvested at the indicated times following the onset of nitrogen starva-tion(?N).(B)Quantitation of results from panel A for eRF3,eIF4GI, and Pat1p.(C)Quantitation of results from panel A for Dcp2p and Pop2p.For Dcp2p blots,the arrow indicates the Dcp2p band,while the small star indicates a nonspecific band recognized by the HA anti-body.For quantitation,protein abundance was normalized to Pgk1p from the same extract as an internal control.All experiments were car-ried out two or more times with similar results.Bar graphs are plotted as mean±standard deviation.(ND)not detectable.

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decreased during nitrogen starvation (Fig.6A),and the decrease of full-length Ubp3p coincided with the appearance of a fragment that was ～30kDa smaller.Since our Ubp3p construct carried a carboxy-terminal HA epitope tag,this in-dicated that the smaller species lacked the amino-terminal re-gion that includes the Bre5p binding site (Li et al.2007).We found that Ubp3p turnover was reduced in the atg7Δstrain (Fig.6B),as well as following the addition of MG132(Fig.6C).Together,these results indicate that both the autophagy and proteasomal pathways participate in Ubp3p depletion during nitrogen starvation.

Since our results suggest that Ubp3p participates in the proteasomal degradation of Dcp2p and Pop2p,we tested for a genetic interaction between the ubp3Δmutation and the pre1-1mutation of the 20S proteasome.Serial dilutions of WT,ubp3Δ,pre1-1,and ubp3Δ/pre1-1strains were plated

on YPD plates in the absence or presence of 1nM rapamy-cin.Colony size and cell viability of these strains were similar on YPD plates grown at 30°C (Fig.6D,left panel).When YPD plates were incubated at 37°C,we found that the colony size of the pre1-1strain was reduced,while the ubp3Δ/pre1-1double mutant grew more slowly and exhibited poorer viability than either of the single mutants (Fig.6D,center panel).When these strains were tested on plates containing 1nM rapamycin at 30°C,the pre1-1strain exhibited only a slight growth defect (Fig.6D,right panel).The ubp3Δstrain exhibited both reduced viability and slow growth,indicat-ing an enhanced sensitivity to TOR1inhibition as observed previously (Kraft et al.2008;Ossareh-Nazari et al.2010).Finally,the ubp3Δ/pre1-1double mutant exhibited both slow growth and a greater loss of viability than the ubp3Δstrain.These results demonstrate that rapamycin sensitivity of the ubp3Δstrain is exacerbated by the pre1-1mutation,further implicating TOR1in the regulation of proteasomal

function.

FIGURE 5.TOR1inhibition by rapamycin induces proteasomal degra-dation of Dcp2p and Pop2p.(A )Western blots and quantitation of Dcp2p abundance following the addition of 200nM rapamycin (+rap)to a wild-type strain.(B )Western blots and quantitation of Pop2p abundance following the addition of 200nM rapamycin (+rap)to a wild-type strain.For panels A and B ,MG132(100μM)was added where indicated.(C )Western blots and quantitation of Dcp2p abundance following the addition of 200nM rapamycin (+rap)to wild-type and tor1-1strains.(D )Western blots and quantita-tion of Pop2p abundance following the addition of 200nM rapamycin (+rap)to wild-type and tor1-1strains.For Dcp2p blots,the arrow indi-cates the Dcp2p band,while the small star indicates a nonspecific band recognized by the HA antibody.For quantitation,protein abundance was normalized to Pgk1p from the same extract as an internal control.All experiments were carried out two or more times with similar results.Bar graphs are plotted as mean ±standard

deviation.

FIGURE 6.Ubp3p is degraded during nitrogen starvation.(A )Western blots and quantitation of Ubp3p following the onset of nitrogen starva-tion (?N)in a wild-type strain.eRF3is included as a control.(B )Western blots and quantitation of Ubp3p following the onset of nitro-gen starvation (?N)in wild-type and atg7Δstrains.(C )Western blots and quantitation of Ubp3p following the onset of nitrogen starvation (?N)in a wild-type strain.MG132(100μM)was added where indicat-ed.For panels A –C ,the arrow indicates the location of full-length Ubp3p.The large star is a stable Ubp3p degradation product,while the small star indicates a nonspecific band recognized by the HA anti-body.(D )Evidence of a genetic interaction between the ubp3Δand pre1-1proteasomal mutations.Wild-type,ubp3Δ,pre1-1,and pre1-1/ubp3Δcells were adjusted to a cell density of one A 600unit/mL and spot-ted on YPD plates,or YPD plates with 1ng/mL rapamycin using fivefold serial dilutions at the indicated temperatures.All experiments were car-ried out two or more times with similar results.Bar graphs are plotted as mean ±standard deviation.

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Effect of Ubp3and autophagy on translational efficiency

Translation is severely reduced during nitrogen starvation (Schmelzle and Hall 2000).This is a consequence of TOR1inactivation,which greatly reduces translation initiation on most mRNAs and allows the expression pattern of specific mRNAs to increase in response to nutrient limitation.However,selective autophagy of proteins,including eIF4G and eRF3,could possibly fine-tune translation during nitro-gen starvation.To address this point,we first asked whether autophagy had any influence on the overall translational down-regulation that occurs upon nitrogen starvation by car-rying out polysome analysis in wild-type and atg7Δstrains.We observed a robust level of polysomes during nutrient-rich growth in both strains (Fig.7A)(polysome/monosome

ratios of 2.0).Following nitrogen starvation for 2h,we ob-served a severe reduction in the polysome pools in both strains (polysome/monosome ratios of <0.2).These results demonstrate that the TOR1-mediated down-regulation of translation initiation during nitrogen starvation is the prima-ry means of controlling bulk translation during nitrogen star-vation,and this mechanism was not adversely affected by the loss of autophagy in the atg7Δstrain.However,we cannot ex-clude the possibility that the reduction in ribosome content or the depletion of specific translation factors may alter the translation of specific mRNAs.

We next asked whether the deubiquitinase Ubp3p influ-ences translation termination during growth in nutrient-rich conditions using dual luciferase readthrough reporters.These reporter constructs contain a nonsense codon (or a sense codon as control)inserted in-frame between the Renilla and firefly reading frames (Fig.7B;Grentzmann et al.1998;Keeling et al.2004).This configuration allows us to use the firefly/Renilla ratio as an indicator of the effi-ciency of translation termination.We found that the ubp3Δstrain exhibited 1.5to 2.5-fold more readthrough at nonsense codons than the wild-type strain,although the steady-state level of eRF3did not change (Fig.7C).This suppressor phe-notype suggests that Ubp3p (and by inference,the ubiquitin –proteasome system)influences the efficiency of translation termination during normal growth conditions.

Finally,we explored the effect of autophagy on translation termination during nitrogen starvation.The efficiency of translation termination is determined by the relative abun-dance of release factors,aminoacyl tRNAs,and terminating ribosomes (Betney et al.2010).During nitrogen starvation,the steady-state levels of both ribosomes and eRF3are re-duced.Accordingly,the reduction in eRF3may modulate the efficiency of translation termination during these con-ditions.To test this possibility,we used dual luciferase read-through reporters to compare the relative stop codon readthrough levels in wild-type and atg7Δstrains in nutri-ent-rich and nitrogen-starved conditions.In the wild-type strain,the readthrough observed was modestly reduced (10%–25%)at the three stop codons during nitrogen starva-tion (Fig.7D),indicating that the efficiency of translation ter-mination had marginally increased.This anti-suppressor effect was not observed in the atg7Δstrain,indicating that autophagy (and possibly the reduced level of eRF3)may fine-tune the efficiency of translation termination during ni-trogen starvation.DISCUSSION

In this study,we found that the translation factors eIF4GI and eRF3undergo rapid and selective Ubp3p-dependent degradation by autophagy following the onset of nitrogen starvation (Figs.1D,E,2).eIF4GI was previously shown to be degraded during either nitrogen starvation or rapamycin exposure,although the mechanism of degradation was

not

FIGURE 7.Effects of autophagy and Ubp3p on translation following the onset of nitrogen starvation.(A )Polysome analysis on wild-type and atg7Δstrains under conditions of nitrogen excess (left )and nitrogen starvation (right ).(P/M)Polysome-to-monosome ratio (where mono-some includes the sum of the 40S,60S,and 80S peaks).(B )Schematic of the dual luciferase readthrough reporters.Each reporter contains ei-ther an in-frame nonsense or sense codon between the Renilla and firefly ORFs.(C )Effect of the ubp3Δmutation on the relative efficiency of translation termination and eRF3abundance during growth in nitrogen excess.(D )Effect of nitrogen starvation (?N)on the relative efficiency of translation termination in wild-type and atg7Δstrains.All experi-ments were carried out two or more times with similar results.Bar graphs are plotted as mean ±standard deviation.

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known(Powers and Walter1999;Kuruvilla et al.2001; Gelperin et al.2002;Ramirez-Valle et al.2008).We found that the turnover of eIF4GI and eRF3is highly selective during nitrogen starvation,since no decrease in abundance of the initiation factors eIF2αand eIF5B,or the elongation factor eEF3were observed under these conditions.Previous studies found that two other initiation factors,eIF4E and eIF4A,also remain stable in both yeast and mammalian cells following nutrient limitation or rapamycin addition (Berset et al.1998;Powers and Walter1999;Kuruvilla et al. 2001;Ramirez-Valle et al.2008).While the degradation of eIF4GI and eRF3shares some components of the ribophagy machinery,the turnover rate of these proteins is much faster than ribophagy,and the overall extent of protein turnover is also much greater.These results indicate that selective autophagy of specific translation factors occurs following the onset of nitrogen starvation in a manner that comple-ments the ribophagy process.

The balance between mRNA synthesis and degradation controls the steady-state levels of cellular mRNAs.Regulation of mRNA synthesis rates has long been appreciated as a key mechanism that controls gene expression,while the impor-tance of regulated mRNA decay is generally less appreciated. We found that the degradation of Dcp2p(an mRNA decap-ping factor)and Pop2p(a component of the CCR4/Pop2p mRNA deadenylase complex)during nitrogen starvation was extremely rapid and dependent upon proteasome func-tion(Fig.3)and the ribophagy deubiquitinase,Ubp3p(Fig.

4).Similarly,a previous study found that Ubp3p interacts with the proteasome to stimulate the selective degradation of a protein(Rad4p)to suppress nucleotide excision repair (Mao and Smerdon2010).The degradation we observed was selective,as other proteins involved in mRNA decay, such as Tpa1p and Lsm1p,were stable under the same con-ditions(Fig.1B).Two other proteins,the poly(A)binding protein Pab1p and decapping enhancer,Pat1p,were also de-pleted by autophagy at different rates(Fig.1C–E).When tak-en together,these results indicate that specific translation and RNA metabolism factors are selectively degraded by either the proteasome or autophagy during nitrogen starvation.It is possible that the differential degradation of these proteins helps cells alter their gene expression profile to adapt to star-vation conditions.

Previous studies found that depletion of either eIF4GI or eRF3reduces growth rates and inhibits TOR signaling in mammalian cells(Chauvin et al.2007;Ramirez-Valle et al. 2008).These similar effects suggest that these proteins may share a common function,such as a shared role in translation. In this regard,both eIF4GI and eRF3participate in the forma-tion of mRNA closed-loop structures required for efficient initiation of cap-dependent translation(Amrani et al.2008). Based on our finding that the deubiquitinase Ubp3p and its binding partner Bre5p are required for efficient degradation of eIF4GI and eRF3(Fig.4A,B),the autophagy machinery and the Ubp3p complex may facilitate eRF3and eIF4GI deg-radation in order to reprogram translation initiation during adaptation to nitrogen starvation conditions.

Previous studies have shown that selective autophagy exists in organisms ranging from yeast to mammals.Targets of this process can range from specific proteins to protein aggre-gates,ribosomes,various organelles,and bacteria.While the mechanism of selective autophagy remains poorly under-stood,a role for ubiquitin is a common theme.For example, degradation of various substrates by autophagy in mammali-an cells requires ubiquitination,as well as ubiquitin-binding receptors such as p62(Kraft et al.2010).Similarly,the selec-tive degradation of ribosomes following nitrogen starvation requires the deubiquitinase activity of Ubp3p and its partner, Bre5p(Kraft et al.2008).Recently,the Ubp3p/Bre5p com-plex was shown to interact with both Cdc48p,which plays a role in the proteasomal pathway,as well as Ufd3p,a ubiq-uitin-binding adaptor of Cdc48(Ossareh-Nazari et al.2010). These Ubp3p partners are required for efficient ribophagy but are not required for bulk autophagy.Since inhibition of proteasomal activity does not block ribophagy,it was pro-posed that Cdc48p functions as a molecular platform on which ubiquitinated ribosomes are recognized and deubiqui-tinated before delivery to the vacuolar for degradation.

In the current study,we found that Ubp3p itself is degraded during nitrogen starvation.The reduced steady-state abun-dance of full-length Ubp3p is accompanied by the accumula-tion of a carboxy-terminal fragment that corresponds to the UCH catalytic domain of Ubp3p that lacks the Bre5p binding domain(Fig.6;Li et al.2007).The loss of full-length Ubp3p and appearance of the Ubp3p fragment are influenced by both the proteasome and autophagy,since inhibition of either pathway moderates the decrease in full-length Ubp3p and the appearance of the degradation fragment(Fig.6B,C). These results suggest that Ubp3p,possibly in complex with known binding partners such as Cdc48p and Ufd3p (Ossareh-Nazari et al.2010),may be regulated by the activities of the proteasome and autophagy during nitrogen starvation to limit these degradative processes.Previous studies have de-scribed a process termed Regulated Ubiquitin Proteasome-dependent processing(RUP)to yield polypeptide fragments with biological activity(Rape and Jentsch2004).It is possible that accumulation of a Ubp3p fragment that retains the UCH catalytic domain but lacks the Bre5p binding domain could down-regulate its deubiquitinase activity and possibly allow it to switch functions(Cohen et al.2003;Li et al.2007). Based on this information and our current results,we pro-pose the model presented in Figure8.During nitrogen excess (Fig.8A),ubiquitination of Dcp2p,Pop2p,eRF3,and eIF4GI may be blocked by TOR1activity(step1).Alternatively,these factors may become ubiquitinated,but their Ubp3p-depen-dent turnover is inhibited by TOR1activity(step2). Following the onset of nitrogen starvation(Fig.8B),TOR1 activity is repressed.This allows Dcp2p,Pop2p,eRF3,and eIF4GI to be ubiquitinated and subsequently processed by the Ubp3p complex(consisting of Ubp3p–Cdc48p–Ufd3p) Selective protein depletion during nitrogen starvation

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(Ossareh-Nazari et al.2010).Engagement of the Ubp3p com-plex leads to the transport of Dcp2p and Pop2p to the protea-some,where their degradation occurs (step 3).Similarly,the Ubp3p complex recruits eRF3and eIF4GI into early auto-phagosomes,leading to their subsequent degradation by autophagy (step 4).The selective degradation of Dcp2p,Pop2p,eRF3,and eIF4GI may gradually become limiting as processing and/or turnover depletes Ubp3p (step 5)during prolonged nitrogen starvation.

The ubiquitin –proteasome system was previously shown to be dispensable for both ribophagy and autophagy in yeast (Krick et al.2010;Ossareh-Nazari et al.2010).However,our results suggest that the ubiquitin –proteasome system medi-ates the regulated degradation of selected proteins during nitrogen starvation.We also found that proteasomal degra-dation of Dcp2p and Pop2p is dependent on TOR1inhibi-tion,suggesting that the TOR1pathway regulates a branch of the ubiquitin –proteasome system (Fig.5).Previous results have also suggested interplay between the proteasome and the TOR1signaling pathway.Chan et al.(2000)found that some proteasome mutants are hypersensitive to rapamycin.Furthermore,rapamycin addition promotes proteasome ac-tivation,while proteasome inhibition decreases TOR func-tion in mammalian cells (Jin et al.2009;Ko et al.2011).These results provide strong evidence of crosstalk between the ubiquitin –proteasome and TOR1pathways,and that TOR1activity inversely correlates with proteasome function under some conditions.

A previous study found that proteasome activity plays an important role in maintaining amino acid availability during the early stages of acute amino acid starvation in mammalian cells,while the autophagy system provides amino acids at later times (Vabulas and Hartl 2005).Here,we observed a similar temporal sequence of events for selective protein deg-radation following nitrogen starvation in yeast.Dcp2p and Pop2p were rapidly and selectively depleted by the protea-some,while eIF4GI,eRF3,and Pat1p were degraded more slowly by autophagy.Other translation and mRNA turnover factors were degraded by autophagy at even slower rates that were comparable to the rate of ribosome turnover by autoph-agy.Finally,some factors were completely stable over the same time period.The selective,temporally controlled nature of this process suggests that the turnover of these proteins is not a bulk process that simply occurs to provide amino acids to the starving cell.Instead,the rapid loss of Dcp2p and Pop2p may increase the stability of a specific subset of mRNAs,as previously shown for strains lacking the RNA turnover factors Xrn1p and Dcp1p (He et al.2003).Similarly,depletion of eIF4GI and eRF3could reprogram the translation machinery to facilitate the synthesis of proteins required for survival during nitrogen starvation conditions.Further studies will be required to test these predictions.MATERIALS AND METHODS

Nitrogen starvation assays

Cells were grown to log phase in synthetic dextrose (SD)media and diluted overnight.When cultures reached a cell density of 0.5A 600units/mL,5A 600units of cells were harvested and flash frozen as the time zero sample.The rest of the culture was centrifuged,washed with nitrogen starvation media (SD medium without amino acids or nitrogen)(Difco)and resuspended in the same volume of nitrogen starvation media.Cells were subsequently harvested at the indicated times.Samples were flash frozen and kept at ?80°C until they were processed for Western blotting.In some experiments cells were washed in nitrogen starvation media with 100μM MG132(Caymen Chemical)and resuspended in the same.In other experi-ments,cells were grown to log,a time =0time point was taken,then 200nM rapamycin (LC Laboratories)was added and aliquots taken at the indicated time-points.

Western blotting and antibodies

To harvest proteins for Western blots,5A 600units of cells were add-ed to 100%trichloroacetic acid (TCA)to achieve a final TCA con-centration of 5%.Cells were incubated on ice for 30min,harvested by centrifugation,washed four times with 100%acetone,and dried under vacuum.Cells were then resuspended in 100μL of sodium dodecyl sulfate (SDS)boiling buffer (1%SDS,50mM Tris,pH 7.5,1mM EDTA),and lysed by agitation with glass beads (four cycles,1min each)with 1min on ice between each cycle.Protein concentrations were determined by the Lowry method (Lowry et al.1951).Fifteen to twenty-five micrograms of protein was loaded into each lane of an 8%SDS polyacrylamide gel.Protein was then transferred to an Immobilon-P transfer membrane (Millipore)us-ing a Genie Electrophoretic Transfer system (Idea Scientific).The membranes were blocked in 0.3%Tween 20–phosphate-buffered saline buffer containing 5%nonfat milk.Membranes were incubat-ed in the same buffer with the indicated antibodies or antiserum at their recommended concentrations for 2h at room

temperature.

FIGURE 8.Model for selective turnover of translation and mRNA de-cay factors during nitrogen starvation by the autophagy and proteasomal pathways.(A )Nitrogen excess conditions.(B )Nitrogen starvation con-ditions.See text for details.

Kelly and Bedwell

906

RNA,Vol.21,No.5

When a secondary antibody was required,either Rabbit anti-mouse (MP Biomedical,55436)or Rabbit anti-rat(Abcam,ab6703)sera were used.Bound antibodies were detected using[125I]-protein A (PerkinElmer,NEX146L100UC),and results were visualized using a Storm PhosphorImager(GE Healthcare).A mouse monoclonal antibody to S.cerevisiae Pgk1p(22C5D8;Invitrogen,cat.No. 459250)was used as an internal control.Rabbit antiserum to eIF4GI was a gift from Michael Altmann(Berset et al.1998). Rabbit antisera to S.cerevisiae eEF3,eEF1A,eEF2were gifts from Terry Kinzy,and rabbit antisera to eIF2α,eIF5B,and eIF5A were gifts from Tom Dever.Rabbit antiserum to Tom70p(Mas70p) was published previously(Koh et al.2001),as were rabbit antisera to eRF3or eRF1(Salas-Marco and Bedwell2004).Mouse monoclo-nal antibodies to S.cerevisiae Rpl3p(ScRPL3)were obtained from the Developmental Studies Hybridoma Bank.HA antibodies (Covance,MMS-101R)were used to detect HA-tagged versions of Lsm1p,Pat1p,Dcp2p,Pop2p,and Ubp3p.Pab1p antibodies are available commercially(EnCor Biotech,MCA-1G1).In some cases, nonspecific bands were consistently observed in Western blots using HA antibodies.When these were near proteins of interest(e.g., Dcp2p or Ubp3p),the correct band was confirmed by its absence in extracts from the same strain lacking the HA-tagged protein. Pulse-chase experiments

To measure protein half-life,16A600units of cells were grown to log (0.5A600)in synthetic dextrose(SD)medium overnight at30°C.The culture was then split into two equal portions.Cells were spun down and resuspended in the same prewarmed media to an OD600of4 OD/mL and incubated at30°C for5min in a shaking water bath. EasyTag[35S]protein labeling mix(PerkinElmer)was added to each tube at200μCi/mL final volume and incubated for15min at30°C with shaking.After15min a150×chase mix(1mg/mL me-thionine,1mg/mL cysteine,15%yeast extract)was added to1×final concentration and cells were incubated at30°C for2min in a shak-ing water bath.Cells were then rapidly harvested and washed.One tube was washed in SD medium without methionine,while the oth-er was washed in SD medium without nitrogen or amino acids.Cells were spun down once more and resuspended in the same wash me-dium.Time-points were taken at the indicated times and flash fro-zen.Samples were incubated with5%TCA and washed twice with ice-cold acetone.The samples were then dried,resuspended in50μL SDS boiling buffer,disrupted by glass bead lysis,and subjected to immunoprecipitation as previously described(Bedwell et al. 1987).A rabbit antiserum to Tom70p(Mas70p)was used as an in-ternal control for immunoprecipitation samples(Koh et al.2001). The final samples were subjected to8%SDS-PAGE,and the gel was dried onto filter paper.Radiolabeled proteins were detected us-ing a Storm PhosphorImager(GE Healthcare).

Polysome profiles

Polysome profiles were conducted as described(Landry et al.2009). Briefly,strains were grown in SD medium to a cell density of0.5A600 units/mL.Cycloheximide was added to a final concentration of0.1 mg/mL,and cells were harvested by centrifugation(6000rpm,4min at4°C)and washed twice with lysis buffer(20mM Tris–HCl at pH 8.0,140mM KCl1.5mM MgCl2,0.5mM DTT,1%Triton X-100, 0.1mg/mL cycloheximide,1mg/mL heparin).After centrifugation (7000rpm for4min,4°C),pellets were resuspended in lysis buffer and cells were lysed by glass bead beating.Lysates were cleared by centrifugation at9500rpm for5min at4°C and layered over a 20%–50%sucrose gradient(containing20mM Tris–HCl at pH 8.0,140mM KCl,5mM MgCl2,0.5mM DTT,0.1mg/mL

TABLE1.Strains and plasmids used

Strain Description Reference

YDB0646MATa leu2-3,112his3-11,15trp1-1ura3-1ade1-14pop2::TRP1PSI–Keeling et al.(2006) YDB0686MATa leu2-3,112his3-11,15trp1-1ura3-1ade1-14pop2::TRP1PSI–atg7Δ::HIS3pDB1227This study

YDB0679MATa leu2-3,112his3-11,15trp1-1ura3-1ade1-14pop2::TRP1PSI–ubp3Δ::LEU2pDB1227This study

YDB0680MATαhis3-Δ200;leu2-3,112;ura3-52;lys2-801;trp1-1;atg7Δ::TRP1DCP2-HA::HIS3This study

YDB0681MATαhis3-Δ200;leu2-3,112;ura3-52;lys2-801;trp1-1DCP2-HA::TRP1This study

YDB0682MATαhis3-Δ200;leu2-3,112;ura3-52;lys2-801;trp1-1;ubp3Δ::HIS3DCP2HA::TRP1This study

YDB0683MATαhis3-Δ200;leu2-3,112;ura3-52;lys2-801;trp1-1;ubp3Δ::HIS3PAT1-HA::TRP1This study

YDB0687MATαhis3-Δ200;leu2-3,112;ura3-52;lys2-801;trp1-1;UBP3-HA3::TRP1This study

YDB0688MATαhis3-Δ200;leu2-3,112;ura3-52;lys2-801;trp1-1PAT1-HA::TRP1This study

YDB0665MATαhis3-Δ200;leu2-3,112;ura3-52;lys2-801;trp1-1;atg7Δ::TRP1This study

YDB0689MATαhis3-Δ200;leu2-3,112;ura3-52;lys2-801;trp1-1;UBP3-HA3::TRP1;atg7Δ::LEU2This study

YDB0695MATa pre1-1pre2-2ura3leu2-3,112his3-11,15Can s Gal+DCP2-HA::HIS3This study

YDB0694MATa ura3leu2-3,112his3-11,15Can s Gal+DCP2-HA::HIS3

MHY501MATαhis3-Δ200;leu2-3,112;ura3-52;lys2-801;trp1-1Amerik et al.(2000) MHY659MATαhis3-Δ200;leu2-3,112;ura3-52;lys2-801;trp1-1;ubp3Δ::HIS3Amerik et al.(2000) YDB0717MATαhis3-Δ200;leu2-3,112;ura3-52;lys2-801;trp1-1eIF4GI-HA::TRP1This study

YH129/w MAT a ura3leu2-3,112his3-1,15can S Gal+Dieter Wolf

YH129/1MATαura3leu2-3,112his3-1,15can S Gal+pre1-1Dieter Wolf

YDB0708MAT a ura3leu2-3,112his3-1,15canS Gal+ubp3Δ::HIS3This study

YDB0709MATαura3leu2-3,112his3-1,15canS Gal+pre1-1ubp3Δ::HIS3This study

YWO607MATa ura3leu2-3,112his3-11,15Can s Gal+Dieter Wolf

YWO612MATa pre1-1pre2-2ura3leu2-3,112his3-11,15Can s Gal+Dieter Wolf

yJC750MATa,ura3,leu2,his3,met15,LSM1-HA::HIS3Jeff Coller

Selective protein depletion during nitrogen starvation

https://www.wendangku.net/doc/8510042541.html,907

cycloheximide,1mg/mL heparin).Gradients were centrifuged in a Beckman SW41rotor at35K rpm for160min at4°C.Fractions were collected,and absorbance at254nm was recorded using an ISCO UA-5absorbance monitor(Teledyne).

Strains

The Saccharomyces cerevisiae strains used in this study are described in Table1.Strains YDB0665,YDB0657,and YDB0707were derived from strain MHY501using standard genetic techniques.Strains YDB0681,YDB0680,YDB0682,YDB0688,YDB0695,YDB0683, YDB0687,YDB0689,and YDB0717were derived from strains MHY501,MHY659,or YDB0665by integrating carboxy-terminal tags into the appropriate genomic locus as described(Longtine et al.1998).YDB0686and YDB0679were made using standard ge-netic techniques starting with strain YDB0646/pDB1227.Strains YDB0708and YDB0709were derived from YH129/w and YH129/ 1,respectively.Strains YDB0694and YDB0695were derived from YWO607and YWO612as described(Longtine et al.1998). yJC750was a gift from Dr.Jeff Coller.YJW615was a gift from Dr.Jonathan Weissman and YH129/w,YH129/1,YWO607,and YWO612were gifts from Dr.Dieter Wolf.

Plasmids

The plasmids used in this study are described

in Table2,while the oligonucleotides used

are shown in Table3.pDB1227was made by

first amplifying a SalI-POP2-HA-BamHI frag-

ment from chromosomal DNA using primers

DB2996and DB2997,and inserting the result-

ing fragment into the SalI–BamHI sites of

YCpLac33(Gietz and Sugino1988).A

HindIII–SalI fragment corresponding to the

500bp preceding the start codon was then am-

plified from chromosome DNA using primers

DB2998and DB2999was inserted into the

HindIII–SalI https://www.wendangku.net/doc/8510042541.html,stly,a fragment corre-

sponding to248bp distal to the stop codon

was amplified using primers DB3000and

DB3001from chromosome DNA and inserted

into the BamH1–SacI sites of the construct.

The final construct was confirmed by se-quencing using primers DB3023,DB3024,and DB3025.pDB1226 was made by first amplifying the TPA1ORF932bp upstream start and200bp downstream stop codon with primers DB0873and DB0874and inserting this into the PstI–BamHI sites of YCpLac22 (Gietz and Sugino1988).A SalI restriction site was then introduced at the stop codon using site-directed mutagenesis and primers DB0876and DB0877.The amplified fragment was subcloned back into the original construct using the KpnI and BamH1sites. Finally,a～250bp SalI–BamHI fragment with a SalI site followed by an HA tag and200bp of the TPA13′UTR followed by a BamHI site was amplified with DB0874and DB3184and inserted into the SalI–BamH1sites of the construct.For pDB1287,the5′UTR and ORF of SCH9was amplified and HA-tagged using primers 3933and3934from chromosomal DNA giving a～3000bp fragment with HindIII–BamHI sites.This was cloned into the HindIII–BamHI sites of YCplac33(Gietz and Sugino1988).The500bp3′UTR was amplified using primers3935and3936to introduce BamHI and SacI sites.This was then cloned into the larger construct.Finally, plasmid pYDF23,which carries the dominant-negative tor1-1allele, was a kind gift from Dr.Ted Powers.

Dual luciferase assays

The dual luciferase reporters used to monitor readthrough of stop codons in yeast were described previously(Keeling et al.2004). Strains harboring the indicated plasmids were grown to log in selec-tive synthetic media and assayed for readthrough either during nu-trient excess or6h after the onset of nitrogen starvation.The dual luciferase assays were carried out as previously described (Grentzmann et al.1998;Keeling et al.2004).This system monitors readthrough of a stop codon by measuring firefly luciferase activity, and allows the normalization to the level of upstream Renilla lucif-erase activity expressed in the same open reading frame.Assays were also done with reporter that contained a sense codon in place of the stop codon to determine maximum(100%)readthrough. Serial dilution assays

Cells were grown overnight in YPD.Of note,1A600unit of cells was spun down and resuspended in1mL sterile water.Fivefold serial

TABLE2.Plasmids used

Plasmid Description Reference

pDB1227POP2-HA/YCplac33This study

pDB1226TPA1-HA/YCpLac22This study

pDB0690CTY775/luc CGAC Keeling et al.(2004)

pDB0691CTY775/luc UGAC Keeling et al.(2004)

pDB0722CTY775/luc CAAC Keeling et al.(2004)

pDB0723CTY775/luc UAAC Keeling et al.(2004)

pDB0720CTY775/luc UAGC Keeling et al.(2004)

pDB0721CTY775/luc CAGC Keeling et al.(2004)

pDB0688CTY775/luc CAAA Keeling et al.(2004)

pDB0689CTY775/luc UAAA Keeling et al.(2004)

pYDF23LEU2,CEN/ARS tor1-1Reinke et al.(2006)

TABLE3.Oligonucleotides used

Oligonucleotide Sequence

DB08735′-CCGGCTGCAGATCAAGAATGCTAATCAATTC-3′

DB08745′-CCGGGGATCCAGTTAAACTTATATTCATTC-3′

DB08765′-GGAAGATGAAGCGTCGACAATTAACCCGTC-3′

DB08775′-GACGGGTTAATTGTCGACGCTTCATCTTCC-3′

DB29965′-GGCCGTCGACATGCAATCTATGAATGTACA-3′

DB29975′-GGCCGGATCCTTATTATGCGTAATCCGGCAC

GTCGTAGGGATATTGGTCCCCATCAATACCGT-3′

DB29985′-GGCCAAGCTTGAAGAAAGAAGTTGAGAAGA-3′

DB29995′-GGCCGTCGACAATCCTTTTTGACCCTTTAT-3′

DB30005′-GGCCGGATCCTCATTTATGTACTATATGTA-3′

DB30015′-GGCCGAGCTCTCTTTCATTAATCGCCACCT-3′

DB31845′-GGCCGTCGACGTATCCCTACGACGTGCCGG

ATTACGCATAAACAATTAACCCGTCTTATTA-3′Kelly and Bedwell

908RNA,Vol.21,No.5

dilutions were made in sterile water and3μL of each dilution was spotted onto YPD plates in the absence or presence of1ng/mL rapa-mycin.Plates were incubated as indicated.

ACKNOWLEDGMENTS

The authors thank Jeff Coller,Jonathan Weissman,Roy Parker,Ted Powers,and Dieter Wolfe for providing strains and plasmids,and Michael Altmann,Terri Kinzy,and Tom Devor for providing anti-sera.The authors also thank Kim Keeling for providing critical com-ments on the manuscript.This work was supported by National Institutes of Health(NIH)R01grant GM068854(to D.M.B.). Received March12,2014;accepted January5,2015.

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Kelly and Bedwell

910RNA,Vol.21,No.5

10.1261/rna.045211.114Access the most recent version at doi: 2015 21: 898-910 originally published online March 20, 2015RNA

Shane P. Kelly and David M. Bedwell

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Saccharomyces turnover factors during nitrogen starvation in Ubp3p-dependent depletion of a subset of translation and RNA Both the autophagy and proteasomal pathways facilitate the References

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核糖体与核酶知识

1. 核糖体(riboso me) 核糖体是细胞内一种核糖核蛋白颗粒(ribonucleoprotein particle), 其惟一功能是按照mRNA的指令将氨基酸合成蛋白质多肽链,所以核糖体是细胞内蛋白质合成的分子机器。 按核糖体存在的部位可分为三种类型:细胞质核糖体、线粒体核糖体、叶绿体核糖体。按存在的生物类型可分为两种类型：真核生物核糖体和原核生物核糖体。原核细胞的核糖体较小, 沉降系数为70S，相对分子质量为2.5x103 kDa,由50S和30S两个亚基组成; 而真核细胞的核糖体体积较大, 沉降系数是80S，相对分子质量为3.9～4.5x103 kDa, 由60S和40S两个亚基组成。 在真核细胞中, 核糖体进行蛋白质合成时，既可以游离在细胞质中, 称为游离核糖体, 也可以附着在内质网的表面, 称为膜旁核糖体或附着核糖体。真核细胞含有较多的核糖体, 每个细胞平均有106～107个, 而原核细胞中核糖体较少每个细胞平均只有15×102～18×103个。 典型的原核生物大肠杆菌核糖体是由50S大亚基和30S小亚基组成的。在完整的核糖体中，rRNA约占2/3, 蛋白质约为1/3。50S大亚基含有34种不同的蛋白质和两种RNA分子，相对分子质量大的rRNA的沉降系数为23S，相对分子质量小的rRNA为5S。30S小亚基含有21种蛋白质和一个16S的rRNA分子。 真核细胞核糖体的沉降系数为80S，大亚基为60S，小亚基为40S。在大亚基中，有大约49种蛋白质，另外有三种rRNA∶28S rRNA、5S rRNA 和5.8S rRNA。小亚基含有大约33种蛋白质，一种18S的rRNA。 2. 基因扩增(gene a mp li fica tion) 细胞内选择性复制DNA, 产生大量的拷贝。如两栖类卵母细胞在发育的早期，rRNA基因的数量扩增到1000多倍。基因扩增是通过形成几千个核进行的，每个核里含有几百拷贝的编码28S、18S和5.8S的rRNA基因，最后卵母细胞中的这些rRNA基因的拷贝数几乎达到50万个，而在相同生物的其它类型细胞中，这些rRNA基因的拷贝数只有几百个。卵母细胞中有如此众多的rRNA基因拷贝，为卵细胞在受精后的发育过程中合成大量核糖体创造了条件。 至于卵母细胞中rRNA基因扩增的机制，有人认为可能是通过从染色体上分离出来的环状DNA分子，这种环状DNA中含有rRNA基因，但是第一个含有rRNA基因的环状DNA是如何形成的尚不清楚。由于环状DNA 能够通过滚环复制(rolling circle replication)的方式进行复制，因而能够产生大量的rRNA基因。 3. 5S rRNA基因(5S rRNAgene)

细胞免疫学论文

【摘要】作为一种具有靶向性的生物大分子，单克隆抗体始终是人们关注 的热点之一，被广泛用于治疗肿瘤、病毒感染和抗移植排斥等。但鼠源单克隆 抗体的临床应用受限于诱导产生人抗鼠抗体、肿瘤渗入量低、亲和力低和半衰 期短等。随着分子生物学技术的发展及其向各学科的渗透，通过基因操作技术 对抗体进行改造，可使其适用于多种疾病的治疗。抗体人源化已经成为治疗性 抗体的发展趋势，同时各种抗体衍生物也不断涌现，它们从不同角度克服了抗 体本身的应用局限，也为治疗人类疾病提供了利器。本文简要介绍上述技术的 基本原理、特点和治疗性抗体的研究进展。 【关键词】人--鼠嵌合抗体生物导弹人源化抗体双特异性抗体 【正文】 一、治疗性抗体技术的研究背景 2000年前，人们将自白喉杆菌培养上清液中分离到的可溶性毒素注入马体内，发现得到的抗血清可以治疗白喉，这是第一个用抗体治疗疾病的例子。随 着免疫学和分子生物学技术的发展，以及抗体基因结构的阐明，DNA 重组技术 开始被用于抗体的改造，人们可以根据需要对以往的鼠抗体进行相应的改造， 以消除抗体应用的不利性状或增加新的生物学功能，还可用新的技术重新制备 各种形式的重组抗体，标志着基因工程抗体时代的来临。自第一个基因工程抗体———人--鼠嵌合抗体于1984 年诞生以来，新型基因工程抗体不断出现，包括人源化抗体、单价小分子抗体（Fab、单链抗体、单域抗体等）、多价小分子 抗体（双链抗体、三链抗体、微型抗体等）、某些特殊类型的抗体（双特异抗体、抗原化抗体、细胞内抗体等）及抗体融合蛋白（免疫毒素、免疫黏连素等）等。用于制备新型抗体的噬菌体抗体库技术成为继杂交瘤技术之后生命科学研究中 又一突破性进展。在噬菌体抗体库的基础上，近年来又发展了核糖体展示抗体 库技术，利用核糖体展示技术筛选抗体的整个过程均在体外进行，不经过大肠 杆菌转化步骤，因此可以构建高容量、高质量的抗体库，更易于筛选高亲和力 抗体和利用体外进行的方法对抗体性状进行改造，核糖体展示抗体库技术代表 了抗体工程的未来发展趋势。 二、各种抗体治疗作用的机理与应用 2.1 抗体的基本组成 抗体的基本单位是由4 条肽链组成的对称结构，包括2 条相同的重链和2 条相同的轻链。重链和轻链分别由可变区和恒定区组成。可变区中的互补决定区与抗体和抗原结合的多样性直接有关，而恒定区的结构与抗体的生物学活性 相关。在少数情况下，抗体与抗原结合后可以对机体直接起保护作用，如用抗 体中和毒素的毒性，但在多数情况下需要通过效应功能灭活或清除外来抗原。

高级分子遗传学复习提纲

高级分子遗传学复习题 1、概念解释： PDT 噬菌体展示技术（phage displayed technology，PDT）是将外源蛋白或多肽与噬菌体外壳蛋白融合，展示在噬菌体表面并保持特定的空间构象，利用特异性亲和作用以筛选特异性蛋白或多肽的一项新技术。该技术将基因型与表型、分子结合活性与噬菌体的可扩增性结合在一起，是一种高效的筛选新技术。目前已成功应用于抗原表位分析，单抗筛选，蛋白质功能拮抗多肽或模拟多肽的确定等。 DNA shuffling 将不同品系具有不同突变位点的基因(1～6kb)或同一家族的基因混合，用DNase I酶切构成随机DNA 片段库(Pool)。用此库样品为模板、以小分子引物进行PCR扩增，一些随机模板得到扩增，由于片段间存在同源性，在退火过程中常出现模板转换(switch)，从而有可能出现集多种突变点于一个基因上的DNA分子，可从多种多样的重组分子中筛选出有用基因。 卫星RNA(satellite RNA) 类病毒(viroids)和拟病毒(virusoids)中类病毒是有侵染性并能独立作用的RNA分子，没有任何蛋白质外壳。拟病毒在构成上与类病毒类似，但是被植物病毒包装，与一个病毒基因组包被在一起。拟病毒不能独立复制，需要病毒帮助其复制。有时拟病毒又称为卫星RNA(satellite RNA)。 交换固定(crossover fixation) 指某一基因簇中的突变通过不等交换趋向扩展到整个基因簇的现象。结果突变的基因要么被淘汰，要么占据全部原来相同基因的位置。 分子伴侣(chaperone) 一种能诱导靶蛋白质形成特定构象使其正确组装的蛋白质。 空转反应(idling reaction) 当空载tRNA进入A位点时,核糖体产生pppGpp 和ppGpp, 诱发应急型反应。 AARS：（氨酰-tRNA合成酶） 催化氨基酸和tRNA2‘或3’－OH共价连接的酶。根据氨基酸序列，可将AARS分为I、II型两组。I 型：Arg、Gln、Glu、Ile、Leu、Trp、Tyr、Val、Cys-RS，其余为II型。I 型RS含有HIGH签名序列(His-Ile-Gly-His)和KMSKS(Lys-Met-Ser-Lys-Ser)序列，使AA结合在3'A的2'-OH上，可以在2'、3'之间移动。II型RS无签名序列，而有3个保守基序。 RNAi/RNAq（RNA干扰、RNA压制） 转录后基因沉默广泛存在于各种生物中，在植物中被称为转录后基因沉默(PTGS)，在动物中被称为RNA 干扰(RNA interference, RNAi)，在真菌中则被称为RNA压制(RNA quelling，RNAq)。尽管叫法不同，但都具有相似机制，都启动一种特殊的RNA降解过程。 酸性面条(negative noodle)

核糖体带动抗生素研究

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人源化抗体的研究进展 摘要：单克隆抗体的问世使得人们对于一种新的治疗疾病的药物充满期待，然而鼠源性抗体往往会受到人体免疫系统的排斥，因而抗体的人源化已成为治疗性抗体的发展趋势。用人抗体取代鼠抗体，是克服鼠单抗临床应用障碍的关键。随着分子生物学研究的深入和一些技术的突破，抗体人源化技术日益成熟。大量人源化抗体已经被广泛应用于临床试验和应用。本文主要介绍了目前人源化抗体构建的三种方法：嵌合、重构和表面重塑，并对人源化抗体的未来发展趋势进行了展望。 关键字：基因工程抗体人源化 1 基因工程抗体简介 基因工程抗体(genetically engineered antibod2ies ，GEAb)是按人工设计所重新组装的新型抗体分子，它既保留或增加了天然抗体的特异性和生物学活性，又去除或减少了无关结构，降低或基本消除抗体的免疫原性，使抗体人源化，并改善抗体的药物动力学，具有生产简单，价格低廉，容易获得稀有抗体的优点，具有广阔的临床应用前景。其主要技术原理是：首先从杂交瘤或免疫脾细胞、外周血淋巴细胞等提取mRNA，逆转录成cDNA，再经PCR分别扩增出抗体的重链及轻链基因，按一定的方式将两者连接克隆到表达载体中，并在适当的宿主细胞(如大肠杆菌、CHO细胞、酵母细胞、植物细胞及昆虫细胞等)中表达并折叠成有功能的抗体分子，筛选出高表达细胞株，再用亲和层折等手段纯化抗体片段[1]。 1984年，Morrison等首次报道人鼠嵌合抗体在骨髓瘤成功表达，标志着基因工程抗体的诞生。1986年，Jones等人源化抗体构建和表达成功。1988年，Skerra 等第一次证明抗体的F ab和F v片段可以在大肠杆菌(E。coli)中正确地装配成保持原抗体特异性的小分子抗体。1989年，Huse等用外分泌型载体构建成功小鼠抗体库，利用抗体库技术获得了全人源化的抗体。1994年，德国基因工程抗体研究小组成功地将基因工程抗体在培养细胞中表达，抗体释放到组织培养液中，获得了较高的抗体产量[2]。 抗体药物的最大特征在于它识别抗原的高度专一性。本文主要介绍人源化抗体的发展历程与研究进展。近几年来随着鼠单抗人源化技术越来越成熟大量的人源性单抗被用于临床治疗肿瘤研究，并取得一定进展，由于其具有高效、低毒、病人不易产生抗药性等优点，同时又克服鼠单抗半衰期短、反复应用会引进病人的等缺点，人源性单抗已成为继手术切除、放疗及化疗后又一治疗肿瘤的药物[3]。 2 人源化抗体的发展 早在一个世纪前，Paul Ehrlich就把抗体形容为“魔弹”，1975年杂交瘤技术建立以后，大量制备含有相同抗原决定簇的单克隆抗体成为可能，从而使“魔弹”进入了临床试验阶段[4]。1982年，当Philip Karr将第一株抗独特型单抗(anti-1d)应用于B细胞淋巴瘤的临床治疗并取得成功之后[5]，治疗性抗体的研究很快成为

核糖体习题

第十章核糖体 本章目标 1.掌握核糖体的种类，形态结构及生理功能。 2.掌握蛋白质合成的基本过程。 3. 一、选择题 （一）A型题 1．细胞中合成蛋白质的场所是 A．溶酶体B．滑面内质网C．细胞核D．核糖体E．细胞质 2．游离于细胞质中的核糖体，主要合成 A．外输性蛋白质B．溶酶体内蛋白C．细胞本身所需的结构蛋白 D．膜骨架蛋白E．细胞外基质的蛋白质 3．组成核糖体的核糖核酸为 A．mRNA B．tRNA C．rRNA D．sRNA E．以上都不是 4．真核细胞质中核糖体的大小亚基分别为60S和40S，其完整的核糖体颗粒为A．100S B．80S C．70S D．120S E．90S 5．下列哪一结构中不含核糖体 A．细菌B．线粒体C．精子D．癌细胞E．神经细胞 6．在蛋白质合成的过程中，肽键的形成是在核糖体的哪一部位 A．供体部位B．受体部位C．肽基转移酶位D．GTP酶活性部位 E．小亚基 7．肽基转移酶存在于 A．核糖体的大亚基中B．核糖体的小亚基中C．mRNA分子内 D．tRNA分子内E．细胞质中 8．核糖体小亚基结合到mRNA上时，所需要的起始因子是 A．IF l B．IF2C．IF3D．Tu E．Ts 9．在蛋白质合成的过程中，氨酰tRNA进入核糖体的哪一部位 A．供体部位B．受体部位C．肽转移酶中心D．GTP酶部位 E．以上都不是 10．在蛋白质合成过程中，tRNA的功能是 A．提供合成的场所B．起合成模板的作用C．提供能量来源 D．与tRNA的反密码相识别E．运输氨基酸 11．真核细胞核糖体小亚基中所含rRNA的大小为 A．28S B．23S C．18S D．16S E．5S 12．在蛋白质合成过程中，mRNA的功能是 A．起串连核糖体作用B．起合成模板的作用C．起激活因子作用D．识别反密码E．起延伸肽链作用 13．肝细胞合成血浆蛋白的结构是 A．线粒体B．粗面内质网C．高尔基复合体D．核糖体E．扁平囊泡

答案-- 9.核糖体

第九章核糖体 一、填空题 1. 真核生物有三种RNA聚合酶，其中聚合酶Ⅲ转录。 tRNA 2. 原核和真核生物的mRNA至少有三种差别：①； ②；③。 真核生物mRNA有5‘帽子结构;3’有poly（A）结构；原核的mRNA是多顺反子3. 组成真核生物核糖体大亚基的rRNA有三种，分别是、、。 18S 5.8S 28S 4. 原核生物和真核生物的核糖体分别是70S和80S，而叶绿体的核糖体是，线粒体的核糖体则是。 70S 55S 5. 在蛋白质合成过程中，mRNA是蛋白质合成的，tRNA 是按密码子转运氨基酸的，而核糖体则是蛋白质合成的。 模板运载工具装配场所 6．真核生物有三种RNA聚合酶，分别转录不同的基因，如RNA聚合酶Ⅰ转录。 rRNA 7．核糖体是一种可以进行自我组装的细胞器。真核生物的核糖体是在细胞核内装配的。编码三种rRNA的基因在染色体上属于同一个，并在核仁中转录成的前体。离体实验表明：原核生物核糖体30S小亚基上的21种蛋白质中，有种是初级结合蛋白，是直接与的rRNA结合的；剩下的次级结合蛋白并不直接与rRNA结合，但它们是维持核糖体功能所必需的。 18S、5.8S、28S 转录单位45S的rRNA 14 16S 二、判断题 1．原核生物和真核生物的核糖体都是在胞质溶胶中装配的。 错。真核生物的核糖体是在核仁中装配的。 2．原核生物和真核生物核糖体的亚基虽然不同，但两者形成的杂交核糖体仍能进行蛋白质合成。 对。 3．细胞内一种蛋白质总量是否处于稳定状态，取决于其合成速率、催化活性以及降解速率。 错。蛋白质的含量取决于合成和降解的比率，而与催化活性无关。4．mRNA的合成是从DNA模板链的3，末端向5‘末端方向移动进行，而翻译过程则是从mRNA模板的5’末端想3‘末端进行。 对。 5．氯霉素是一种蛋白质合成抑制剂，可抑制细胞质核糖体上的蛋白质合成。 错。它只能抑制70S核糖体进行蛋白质合成，而不能抑制80S核糖体进行蛋白质合成。 6．单个核糖体的大小亚基总是结合在一起，核糖体之间从不交换亚基。 错。在每一轮翻译后，核糖体的亚基之间会进行互换。当核糖体从一条mRNA 链上释放下来后，它的两个亚基解体，进入一个含游离大亚基和小亚基的库，并

3.2细胞内物质的合成

课题第二节细胞内物质的合成 教学重点1.核糖体和内质网的形态、分类和功能 2.物质在细胞内的合成 教学难点物质在细胞内的合成 教学目标知识目标 1.知道核糖体和内质网形态结构、分类、组成成分、分布特点尤其是分类与功 能 2.简述物质在细胞内的合成过程 能力目标 1.学会应用构建生物模型的方法加强对抽象结构和概念的理解 2.培养学生的观察能力、思维能力. 情感目标 1.通过结构与功能相适应的生物学思想,加强对唯物主义、辨证观的树立和培养 2. 通过核糖体和内质网及高尔基体对蛋白质合成的协作,加强同学们对团队的 认识,加强集体意识和合作精神 法制渗透 教学方法讲授法、直观教具法课时安排 1 教学内容教学过程个人教学札记 一、核糖体与内质网的结构导入：植物细胞中的叶绿体能够利用太阳光能，把水和二氧化碳等无机小分子合成淀粉等有机物。同样，细胞还能将从外界吸收来的其他小分子物质，如氨基酸、胆固醇等合成“建造”细胞及生物体的蛋白质、脂质等生物大分子。那么，细胞内的哪些结构能担任此重任呢？生物大分子在细胞内又是怎样合成的呢？今天我们就来学习生物大分子蛋白质和脂质等的合成及运输过程。（出题）第二节细胞内物质的合成 引导学生看细胞结构图片，充分利用教材这个最重要的教学资源和学生的经验，关键要给学生解释清楚很大部分物质是生物细胞合成的！ 1.线粒体是细胞进行有氧呼吸的主要场所。又称＂动力车间＂。细胞生命活动所需的能量，大约95%来自线粒体。 2. 中心体与低等植物细胞、动物细胞有丝分裂有关。 3.核糖体是“生产蛋白质的机器”，有的依附在内质网上称为附着核糖体，有的游离分布在细胞质中称为游离核糖体。 4.内质网是由膜连接而成的网状结构，是细胞内蛋白质的合成和加工，以及脂质合成的“车间”。 5.高尔基体对来自内质网的蛋白质加工，分类和包装的“车间”及“发送站”。 6.溶酶体分解衰老，损伤的细胞器，吞噬并杀死入侵的病毒或细菌。 7.液泡是调节细胞内的环境，使植物细胞保持坚挺的细胞器。含

细胞生物学核糖体的结构及功能

第十一章核糖体 一、核糖体的结构及功能 核糖体是体积较小的无膜包围的细胞器，在光镜下看不到。1958年才把这种含有大量RNA的能合成蛋白质的关键装置定名为核糖核蛋白体ribosome，简称为核糖体。 (一)核糖体的一般性质 1、存在与分布 核糖体存在一切生物的细胞中，包括真核细胞和原核细胞。这是有别于其它细胞器的特点。在真核细胞中，有些核糖体是游离分布在细胞质基质中，也有许多是附着在rER膜及核膜外表。此外，还有核糖体是分布在线粒体和叶绿体的基质中。在原核细胞内，大量核糖体游离在细胞质中，也有的附着在质膜内侧面。细菌的核糖体占总重量的25—30%。 2、形态和大小 一般直径为25—30nm，由大、小两亚单位构成，通常是以大亚单位附在内质网膜或核膜外表。当进行蛋白质合成时，小亚单位先接触mRNA才与大亚单位结合，而合成完毕后又自行解离分开。另外，多个核糖体还可由mRNA串联成多聚核糖体，每个多聚核糖体往往由5-6个核糖体串成，但也有多至50个以上的(例如肌细胞中合成肌球蛋白的多聚核糖体是由60—80个串联而成)。 3.数量和分类 细胞中的核糖体数量多少不一。一般来说，增殖速度快的细胞中偏多，分泌蛋白质的分泌细胞中也较多。例如分泌胆汁的肝细胞中为6×106个，大肠杆菌中为1500—15000个。在不同类型生物细胞之中，核糖体大小及组分都有一定差

异。一般可分为两大类：80S型和70S型。 大亚单位60S 真核生物核糖体80S 小亚单位40S 大亚单位50S 原核生物核糖体70S 小亚单位30S （“S”是沉降系数的衡量单位。大、小亚单位组成核糖体，并非由其两者的S值直接相加，这是因为S值变化其实是与颗粒的体积及形状相关的。） 叶绿体中的核糖体与原核生物的相似，而线粒体中的核糖体则较小且多变，例如哺乳动物的线粒体核糖体是55S，但一般仍将它们都划分到原核生物的70S型。 (二)核糖体的化学组成 主要组分是r蛋白和rRNA，极少或无脂类。70S型核糖体之中，r蛋白: rRNA约1 : 2 ；而在80S型核糖体之中，r蛋白: rRNA约1 : 1 。 核糖 体来源核糖体 大亚 单位 小亚 单位 rRNA r蛋白数量 大亚单位 小亚 单位 大亚 单位 小亚 单位 真核细 胞原核细胞线粒体80S 70S 55S 60S 50S 35S 40S 30S 25S 28S+5S+5.8S 23S+5S 21S+5S 18S 16S 12S 49 31 - 33 21 - 70S和80S型核糖体都含有5S rRNA，其结构大小十分接近，都由120或121个核苷酸组成。这表明古核生物、原核生物和真核生物在进化上的亲缘关系，它是残存在生物体

生物专业英语第三版课文翻译lesson1,4,5

Lesson 1 4 5 Lesson 1 1.细胞质：动态移动工厂 与我们生命相关的大多数性质是细胞质的性质。细胞质大部分由半流体物质组成，并以外部的质膜为界。细胞器悬浮在其中，由丝状的细胞骨架支撑。细胞质中溶解了大量的营养物质，离子，可溶性蛋白以及维持细胞功能的其它物质。 2.细胞核：信息中心 真核细胞的细胞核是最大的细胞器，在染色体上储存着遗传物质（DNA）。（原核细胞的遗传物质存在于拟核中。）细胞核含有一或两个核仁，核仁促进细胞分裂。被穿孔的囊称为核膜，它将细胞核及其内容物从细胞质中分离出来。小分子可以穿过核膜，但较大的分子如mRNA 和核糖体必须通过核孔进入和排出。 3.细胞器：专用的功能单位 所有的真核细胞都含有多种细胞器，每个细胞器在细胞中都有其特定功能。本节介绍的细胞器包括核糖体，内质网，高尔基体，液泡，溶酶体，线粒体和植物细胞中的质体（叶绿体）。 一个细胞中核糖体的数量可能从几百到上千不等，这一数量反映了核糖体是氨基酸被组装成蛋白质以供输出或用于细胞过程的场所这个事实。一个完整的核糖体由一个较大的亚基和一个较小的亚基组成。在蛋白质合成过程中，两个亚基沿着一条mRNA链移动，“读取”编码在其中的基因序列，并将该序列翻译成脯氨酸。多个核糖体可能附着在单个mRNA链上，这种组合被称为多聚体。大多数细胞蛋白质是在细胞质中的核糖体上制造的。可输出的蛋白质和膜蛋白通常在内质网的帮助下产生。 内质网，是一些不规则排列的膜囊，小管，和液泡组成的，可能有光滑和粗糙的区别。两种类型都与蛋白质的合成和运输有关。粗糙内质网上分布着许多核糖体，也可能细胞分裂后核膜的来源。 光滑的内质网上没有核糖体，主要作用是脂肪和类固醇的合成以及细胞内有毒物质的氧化。两种类型的内质网都充当细胞内的隔室，其中特定的产物可以被分离并随后分流到细胞内或细胞外的特定区域。 运输小泡能够将可运输分子从内质网运输到另一个膜质细胞器上。在高尔基复合体内，蛋白质分子被修饰和包装，以输出细胞或运送到细胞质中的其他地方。 细胞中的液泡似乎是中空的，但实际上充满了流体和可溶性分子。最典型的液泡出现在植物细胞中，用作贮水场所和糖以及其他分子的贮存地点。动物细胞中的液泡进行吞噬作用（颗粒物质的摄入）和胞饮作用（空泡饮酒vacuolar drinking）。 液泡的一个亚单位是被称为溶酶体的细胞器，它含有消化酶（包装在高尔基复合体中的溶酶体），可以分解大部分生物大分子。它们起到消化食物颗粒和降解受损细胞部分的作用。 线粒体是所有细胞中产生能量的化学反应的场所。此外，植物细胞含有质体，它们利用光能在光合作用过程中制造碳水化合物。在线粒体内嵴上提供了很大的表面积分布着ATP酶。

抗体药物地研究现状和发展趋势

抗体药物的研究现状和发展趋势 一、研究现状 1．抗体研究发展历程 抗体作为药物用于人类疾病的治疗拥有很长历史。但整个抗体药物的发展却并非一帆风顺，而是在曲折中前进。第一代抗体药物源于动物多价抗血清，主要用于一些细菌感染性疾病的早期被动免疫治疗。虽然具有一定的疗效，但异源性蛋白引起的较强的人体免疫反应限制了这类药物的应用，因而逐渐被抗生素类药物所代替。第二代抗体药物是利用杂交瘤技术制备的单克隆抗体及其衍生物。单克隆抗体由于具有良好的均一性和高度的特异性，因而在实验研究和疾病诊断中得到了广泛应用。 单抗最早被用于疾病治疗是在1982年，美国斯坦福医学中心Levy等人利用制备的抗独特型单抗治疗B细胞淋巴瘤，治疗后患者病情缓解，瘤体消失，这使人们对抗体药物产生了极大的期望。1986年，美国FDA批准了世界上第一个单抗治疗性药物——抗CD3单抗OKT3进入市场，用于器官移植时的抗排斥反应。此时抗体药物的研制和应用达到了顶点。随着使用单抗进行治疗的病例数的增加，鼠单抗用于人体的毒副作用也越来越明显。同时一些抗肿瘤单抗未显示出理想效果。人们的热情开始下降。到20世纪90年代初，抗毒素单抗用于治疗脓毒败血症失败使得抗体药物的研究进入低谷。由于大多数单抗均为鼠源性，在人体反复应用会引起人抗鼠抗体（HAMA）反应，从而降低疗效，甚至可引起过敏反应。因此，一方面在给药途径上改进，如使用片段抗体、交联同位素、局部用药等使鼠源性抗体用量减少，也增强了疗效；另一方面，积极发展基因工程抗体和人源抗体。 近年来，随着免疫学和分子生物学技术的发展以及抗体基因结构的阐明，DNA 重组技术开始用于抗体的改造，人们可以根据需要对以往的鼠抗体进行相应的改造以消除抗体应用不利性状或增加新的生物学功能，还可用新的技术重新制备各种形式的重组抗体。抗体药物的研发进入了第三代，即基因工程抗体时代。与第二代单抗相比，基因工程抗体具有如下优点：①通过基因工程技术的改造，可以降低甚至消除人体对抗体的排斥反应；②基因工程抗体的分子量较小，可以部分

核糖体

1.真核生物有三种RNA聚合酶，其中聚合酶Ⅲ转录。 2.原核和真核生物的mRNA至少有三种差别：①＿；②；③ 3.组成真核生物核糖体大亚基的rRNA有三种，分别是：、、。 4.原核生物和真核生物的核糖体分别是70S和80S，而叶绿体的核糖体是，线粒体的核糖体则是。 5.在蛋白质合成过程中，rRNA是蛋白质合成的，tRNA是按密码子转运氨基酸 的，而核糖体则是蛋白质合成的。 6.细胞核内不能合成蛋白质，因此，构成细胞核的蛋白质（包括酶）主要由合成，并通过引导进入细胞核。 7.RNA编辑是指在的引导下，在水平上改变 8.原核生物线粒体核糖体的两个亚基的沉降系数分别是和。 9.核糖体两个亚基的聚合和解离与Mg2＋浓度有很大的关系，当Mg2＋浓度小于时， 70S 的核糖体要解离；当Mg2＋浓度大于时，两个核糖体聚合成 100S的二聚体。 10.70S核糖体中具有催化活性的RNA是。 11.在蛋白质的合成过程中mRNA起到的作用，即根据mRNA中密码子的指令将合成多肽链中氨基酸按相应顺序连接起来，密码子决定了多肽链合成的起始 位置和其上的氨基酸顺序。然而mRNA的密码子不能直接识别氨基酸，所以氨基酸必须先与相应的tRNA结合形成，才能运到核糖体上。tRNA以其 识别mRNA密码子，将相应的氨基酸转运到核糖体上进行蛋白质合成。因此，通过密码子才能翻译出mRNA上的遗传信息，翻译过程中需要既能携带氨基酸又能识别密码子的tRNA作为连接器，将氨基酸转运到相应密码子的位置，完成蛋白质合成。 12.蛋白酶体既存在于细胞核中，又存在于胞质溶胶中，是溶酶体外的，由10～20个不同的亚基组成结构，显示多种肽酶的活性，能够从碱性、酸性和中性氨基酸的端水解多种与连接的蛋白质底物。蛋白酶体对蛋白质的降解是与环境隔离的。主要降解两种类型的蛋白质：一类是，另一类就是。蛋白酶体对蛋白质的降解通过介导。是由76个氨基酸残基组成的小肽，它的作用主要是识别要被降解的蛋白质，然后将这种蛋白质送入蛋白酶体的圆桶中进行降解。蛋白酶体对蛋白质的降解作用分为两个过程：①对被降解的蛋白质进行标记，由完成；②蛋白酶解作用，由催化。蛋白酶体存在于所有细胞中，其活性受素的调节。

核糖体

第十一章核糖体 选择题 1.组成核糖体的核糖核酸为 A.mRNA B.tRNA C.rRNA D.sRNA 2.真核细胞质中核糖体的大小亚基分别为60S和40S，其完整的核糖体颗粒为 A.100S B.80S C.70S D.90S 3.在蛋白质合成的过程中，肽键的形成是在核糖体的哪一部位 A.供体部位 B.受体部位 C.肽基转移酶位 D.GTP酶活性部位 4.影响核糖体大小亚基结合的金属离子为 A.Ca2+ B.Na+ C.K+ D.Mg2+ 5.肽基转移酶存在于 A.核糖体的大亚基中 B.核糖体的小亚基中 C.mRNA分子内 D.tRNA分子内 6.遗传密码子是指 A.DNA分子上每3个相邻的碱基 B.rRNA分子上每3个相邻的碱基 C.tRNA分子上每3个相邻的碱基 D.mRNA分子上每3个相邻的碱基 7.一个tRNA上的反密码子是UAC，与其相对应的mRNA密码子是 A.CAC B.AUG C.TUG D.ATG 8.以mRNA为模板合成蛋白质的过程称为 A.转录 B.转化 C.翻译 D.复制 9.在蛋白质合成的过程中，氨酰tRNA进入核糖体的哪一部位 A.供体部位 B.受体部位 C.肽转移酶中心 D.GTP酶部位 10.在蛋白质合成过程中，tRNA的功能是 A.提供合成的场所 B.起合成模板的作用 C.与tRNA的反密码相识别 D.运输氨基酸 11.游离于细胞质中的核糖体，主要合成 A.外输性蛋白质 B.溶酶体内蛋白 C.细胞本身所需的结构蛋白 D.高尔基复合体内蛋白 12.参与蛋白质合成的酶是 A.羧基肽酶 B.谷氨酰氨合成酶 C.肽基转移酶 D.连接酶 13.细胞的蛋白合成时，氨基酸活化所需的能源是 A.ATP B.ADP C.GTP D.cAMP 14.真核细胞核糖体小亚基中所含rRNA的大小为

抗体制备方法的研究进展

4 Guy J Hallman,Paisan Loaharanu.Generic Ionizing Rad-i ation Quarantine Treatments Agai nst Fruit Fli es(Diptera: T ephritidae)Proposed.Journal of Economic Entomoloy, 2002,95(5):893~901 5 Peter A Follett and Suzanne S Sanxter.Hot Water Immer-sion to Ens ure Quarantine Securi ty for Cryptophle bia spp. (L epidoptera:Tortricidae)in Lychee and Longan Export-ed from Hawaii.Stored-Product and Uarantine Entomolo-gy,S tord-Product And Uarantine Entomology,2001,94 (5):1292~1295 抗体制备方法的研究进展 邵碧英 (福建出入境检验检疫局 福州 350001) 由于抗体的反应特异性,抗体的应用广泛,不仅是细菌、病毒、外源蛋白等的诊断和检测试剂,而且还是人和动物某些疾病的诊断试剂和有效治疗药物。抗体制备方法经历了常规血清技术、杂交瘤技术、基因工程抗体技术及抗体库技术,本文将分别阐述。 1 常规血清技术 常规的抗体制备包括抗原的制备、免疫动物、抗血清制备及特异抗体纯化等步骤。获得的抗体是针对多个不同抗原决定簇的抗体混合物,因此称之为多克隆抗体,简称多抗。制备抗体,关键是获得高产量、高纯度的抗原。抗原的获得方法有以下几种。 1 1 抗原提取剂 若抗原易获得,或易提纯,则可采用抗原提取剂制备抗体。以制备病原体的抗体为例,若以整个病原体作为抗原,只需对其进行分离、纯化,而以其某部分如菌丝可溶性蛋白、病毒的外壳蛋白等作为抗原,则要采用相应的提取方法。低分子量物质需与大的载体偶联后才可形成完全抗原。陈京等[1]将提纯的番茄环斑病毒、烟草环斑病毒和南芥菜花叶病毒分别免疫兔子,制备的抗血清用于3种病毒的检测。陈松等[2]将从苏云金杆菌菌体中提取的Bt蛋白作为抗原,制备的抗血清用于检测转基因棉花中的Bt蛋白。氯霉素(GAP)是小分子物质,石德时等[3]将GAP 和牛血清白蛋白偶联作为抗原制备了抗血清,用于检测动物性食品中的GAP残留。 1 2 人工合成的多肽 若目的蛋白的氨基酸序列已经清楚或可从相应的核苷酸序列推导,则可合成短肽,与载体蛋白偶联后作为抗原。许家喜等[4]在计算机辅助下根据外源基因cDNA序列预测出蛋白抗原位点肽,人工合成后制成复合抗原,免疫家兔后获得特异性抗体,用于测定转基因植物中外源基因的表达产物。 1 3 基因工程抗原 若病原体难培养或抗原不易提取,则可采用基因工程抗原,制备的一般程序是:克隆抗原基因,构建表达载体,转化受体,使抗原基因得到表达,表达产物经适当纯化即可作为抗原。制备基因工程抗原,关键是选择适当的表达系统。表达系统有以下几种。 1 3 1 原核细胞表达系统:原核细胞表达系统有大肠杆菌、枯草杆菌、链霉菌、蓝细菌等,既可表达原核基因,又可表达真核基因。其中,大肠杆菌最常用,具有经济、易操作、研究和生产周期较短等优点。 1 3 2 真核微生物表达系统:真核微生物表 292 收稿日期:2003-02-20