rough endoplasmic reticular (rer) membrane I I I The second involves the fusion ... membrane has been elusive In particular, the synthesis of TAG has been ...
Biochemical Society Transactions (1997) 25 21s Evidence for overt and latent forms of DGAT in rat liver micrnsomes Implications for the pathways of triacylglgccrnl incorporation into VLDL
Marh OWI:N and Victor A %AMMII' I1ann:ih Research Institute. Ayr, KA6 5111.. Scotland, UK
I lcpatic \a>-Iow-dewity Iipoprokms (Vl,l)l,) acquuc their core tnacylglvccrol (TAG) i n t w steps l'hc lint occui-s during the cotranslational insertion of apol3 through the rough endoplasmic reticular (rer) membrane I I I The second involves the fusion hct\vecn the naxent. relatively lipid-poor pailicles with non-apol3-associated TAG droplets within the lumen ofthe er These droplets are present in the smooth er (ser) iuid fusion with tlie n a m t paiiicles may wcui- at the junction between the ser and the rcr 12 I 'l'he elucidation of the mechanisms involved in 'TAG transfer across the er membrane has been elusive In particular, the synthesis of TAG has been considered to he a pi-cressthat is exclusivel! limited to the outer aspect of the er membrane ever since the work on thc el- toPolop?. of glvccrolipid svnthesis perioimed by Dell and colleagues (see 131) 'Thus it has been proposed that for thc tirst step, the insertion 01' apol3 thmugh the rer membrane -how enables 'TAG to permeate the memhrane and to htvome in.wrted into a hydrophobic pocket tiinned by the folding na.scent polpeptlde. probably b o u g h the involvement of microsomal triacylglycerol transfer protein (MII') which is essential for the assemblv o f I A G into VI,DI. 141 However. the question as to the mechanism through which 'TAG is transferred across the membrane has not heen addressed Similarly, although it has been known for a long tin: that qlosolic droplet TAG is not incorporated en bloc into VI.Dl,-TAG. but that Aist II has t o undergo hydrolysis (see [ j ] ) ,the mechanism whereby the resynthesis of ' T M i leads tu its transmembrane ti-ansferand secretion has not been elucidated It has k n mggebted that the problem represented by the imprimeability of membranes to 'TAG am he oveicomeby Ihe duplication of the entire phosphatidate pathway senes iithesising enqn~cson the lumenal aspect of the er membrane 16 j Ik)wever, two recent developments make it possible to suggest that synthesis of 'TA(i oil the lumenal aspect of the er membrane may he achieved through the bimodal distribution 01' only one enzyme, naniely diacylglycerol acyltransferase (IXiAT) which catalyses the step committed to TAG synthesis. The two sets of obsenratlms m (I) that hydrolysis of cytosolic droplet 1 A G does not proceed fully to its constituent fatly acids, but rather to diacvlglycerols (DAG), which aller remodelling arei~-esterifiedto TAG [7,8], and (ii) that the er membrane contains oveil and latent mediunAong-chain camitine acmansferases 16.91. In view of the well-established ability of D A G to permeate membranes I l O l , the partial hyrolvsis ofcytosolic I A G provides the basis for transfa ofthe glycemyl m i t y across the membrane In addition, by analogy with the camitine aeylyltransferasesystem (if the mitochondria, the sequential actlon ofthe two transferaves in the er would he espected to achieve the transfer ot'acyl mwtlcs across the m b r a n e Consequently, both substrates could be made available fbr DCiAl i f this were also to hc present on the lumenal face ofthc er membrane Therefore, i n the present study we set out to re-evaluate the latency of IXiAI' in microsomal membranes Microsonial fractions were prepared kom 24h-starved male Wistar rats by diIY'=n )mogenates (in 300mM-sucrose. I mM-EGTA and 5mM-'I lion with taurocholate was perlbimed at 0" for 30 min 1 anol acyltransferase activities were measured simulta ning .fOOmM-sucrnse, I OmM-I'm, BmM-MgC1:. 0 XinM yl-sn-glycerol (in 0 2% ethanol). 1 mg/ml defatted albumin. I OOpM- I I"C]-palmitcyl-CoA (22.OOOdpm/nmol) which was sonicated Assays were untiatrd with microsomes and terminated by extraction with chlorofbimmethanol I'rtduets were separated by t.1.c and the radioactivity associated with TAG and ethyl palmitate were quantitated using a phosphorimageiWhen total (unliactionated) microwmes were used, DGAT activity was approsimately doubled alter lvsis of the membrane vesicles with 0.6% taurocholate The Iatcnq oflXiAT was 46.7+6 5% oftotal actlwty (n=9 preparations) As eypected, the activities ofmmose-6-phosphatase and ethanol acyltransferase were highly latent: taunzholate tlcabnent routlnely resulted in approx. IO-fbId increase in their activities In oi-der to asceilain that this etfect was due to lvsis ofthe microsomal membrane rather than :I non-specific efl'ect on LXiAT activiy, dose-i-esponse cuives were o1)t;iined li)r the etl'ecl of increasing taurmholate concenlrations on the latencv ofthe iiativities of IXiA'I and the lumen markers on the same preparations of microsomes. The half-mau;imally eEkX"ectl concenbationof taurocholatc (0.3%) was identical for the e\p)wre 01. a11 hive enzy:s smhed (Fig la) This identity between the concentration dependence of detergent action on the thee activities indicates that the increased IXiAl :icti\ity in the presence of taui-ocholnteresults fi.oni the physical disiuption of the microsonial membrane We conclude fi.om these results that separate DGAT activ~tics(which we designate as I X A T I and 2, respectively), ofthe same order of iniignitude, eyisl on either side of the er menilvane in rat liver. In order to test whether this bimodal distribution of D G A I on the two sides of the cr incnihrane is expressed throughout the er system, we fractionated crude inicrosomes into ser- and rer-ennched fractions on continuous sucrose density gradients (0 37 - I 80-M) M A T activily was measured in intact and taurocholatetreatcd nncrcwnes Allhough the total specilk activity of DGAT was higher in the rer, ~ throughout the gradient (Fig Ib) The the latency 01' the e q m e was v e similar intactness of :ill the fractions was demonstrated hv the high degree of latency of the lumcnal m:irher cmmes
Fig 1(a) I'artral latency of L X A T activit). (*)in rat liver inicrosnines: same tautwholate concentrations nre requiredfor e.rpusure oflutent N A T (Ieb.v-a.ris). niaiwose-6-phosplta.se (.iarid ac.vI-CoA.ethano1ucyltran.fernse (0 ). (b) Latencv oflX~.4~'ocliwt). l/uvug/ioiiIthe er in intact )(. arid /,v.sed (0)niicrusonies .separated on .sucro.~c dewit).puiiierits 10 produce rer- and .ser-enriciiedfructions. Broken line indicare.\ H.Y4 p"fi1,.
I PA
/
er membrane
-
DAG
1-acyl glycerol-3P
7 TAG
protein synthesis
I
,TAG
AcvI.CoA
acvlcarniilnm
I
Fig 2 I'mposed oiwlwnientqf Wi.31' 3 (latent) i n the synthesis oflurnenal TAG,fiw ossocimron with nasceni a.poR (step I / i n ver, and ,fiw forination of non-cipoRassociated intraluinenol1X; droplets i n ser reyuired,for step 2. DGAT I /overt) IS proposed to he involved prinrari!v in tlie finmation of cyrosolic droplet I W . P.4 =p/uisphatidrc acid. CAT, and ( '.17': c,vtosol- and Iunien-Jacing carnitine acy1translera.w ~
Therefore. our data provide evidence for the existence of overt and latent forms o1'lXAT III rat liver i-er and m. We suggest that this bimodal distribution ofthe enzyme obviates the need for the transfer of TAG across the membrane (step I of VLDL core TAG incorporation) or for the duplication of any of the glycerolipid synthesising enzymes (other than DCiAT) on the lumenal aspect of the er memhrane The existence of latent IXA'J 2 would also be able to fulfill the function of Ihe provision ofnon-apoR associated 'TAG within the ser, required for the second step of VL.i)L.-'lAG assembly Therefore, both steps would be able to be achieved through the trand.er across the memhrane of DAG generated either directly from the phosphatidate pathway or from the partial hydrolysis of cylosolic droplet TAG. In the scheme suggested in Fig 2, the cytosol-facing DGAT I would be involved primarily in the synthesis ofcytosolic droplet TAG References
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