ER-associated and proteasomemediated protein degradation: how two topologically restricted events came together

The vast majority of secreted proteins are synthesized on cytosolic ribosomes and translocated into the lumen of the endoplasmic reticulum (ER) either co- or posttranslationallyl. Why do most secreted proteins become compartmentalized inside the cell before being transferred to the extracellular space? While there are probably many answers to this question, it is certain that compartmentalization streamlines processing, folding and monitoring of newly synthesized polypeptides to ensure that only active or properly assembled proteins are secreted. Thus, the ER contains not only enzymes and molecules that facilitate the maturation of a polypeptide but also a quality-control system to prevent immature or abnormal products from escaping the cell. Such defective species are either retained in the ER until they fold correctly or are degraded by a process known as ER-associated protein degradation. This degradation pathway does not exclusively target misfolded or aberrant polypeptides but also regulates the activity of certain ER-resident proteins and the expression of plasma membrane proteins2. The existence of ER-associated protein degradation has been apparent for some time, but the factors required for substrate selectivity and protein quality control, the proteases responsible for degradation and even the site of proteolysis have been difficult to define. In the past year, however, many facets of this process have emerged from divergent studies, lending new insights into how specific proteins are removed from the ER. This review will summarize recently discovered mechanisms of ER-associated protein degradation and speculate on what other factors may be required for efficient degradation in viva Owing to the breadth of research in this field, limitations on space and because excellent reviews on protein degradation exist (e.g. see Refs 2-4), we focus here only on selected examples from the literature.

ER-associated and proteasomemediated protein degradation: how two topologically restricted events came together A protein-degradation

pathway associated with the endoplasmic

reticulum (ER) can selectively remove polypeptides porn the secretory pathway. The mechanisms of this ER-associated protein degradation were obscure, but recent studies using both yeast and mammalian cells have indicated that substrates for degradation are targeted to the cytosol where proteolysis is catalysed by the proteasome. The degradation process is now known to comprise at least three distinct events: first, recognition of a polypeptide for degradation; second, e/j7ux of this substrate ffom the ER to the cytosol; and, finally, degradation by theproteasome.

Common themes degradation

in ER-associated

protein

advances in understanding

This review summarizes recent

how each of these steps is achieved.

While a largenumber of proteins may be targetedfor degradationin the cell, three featuresdefine ER-specific degradation: first, the degraded substratesreside in the ER as judged by immunological or biochemical methods; second, intracellular degradation requires neither lysosomalnor vacuolar hydrolases,assayedby showing that degradation isinsensitive either to weak bases(which raisethe pH of the lysosomeand inactivate resident proteases)or, in yeast, to the disruption of genesencoding vacuolar proteases;and, third, the proteolysis of secretedpolypeptides occursunder conditions in which ER-to-Golgi transport is blocked, demonstrated in yeast by using temperature-sensitive SKmutants or in mammaliancellsby cytosol or energy depletion. By these criteria, at least 25 proteins in eukaryotescan be consideredsubstratesfor ER-associated protein degradatior9. An important aspectof ER-associated protein degradation isthat it is highly selectivefor specificproteins. The proteolysis of monomers unable to associateinto protein complexes in the ER has been investigated extensively and demonstratesthe substrateselectivity of this process.For example, the integral membrane subunits of the T-cell receptor (TCR) assemblein the

Becausethe substratesof this degradation pathway accumulate in the ER and originally were found to be degradedin the absenceof cytoso12,initial studies

trends in CELL BIOLOGY

0 1997 Elsevier Science Ltd. All rights reserved. 0962-8924/97/$17.00

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7) April 1997

Copyright

ERbefore delivery to the plasmamembrane. Three of the subunits, the monomeric forms of the a, p and 6 chains, have very short half lives in viva, whereasthe Eand 5 subunits are stablez. Becausethe a, p and 6 chains becomestableduring complex assembly,they may present recognition motifs to the degradation machinery that become occluded asthe TCR forms. Site-directed mutagenesis has indicated that such motifs may be present in the integral membrane domains of the a and p subunits2- domains that are also required to recognize other subunits in the TCR complex. However, it seemsthat the transmembrane domain alone of the 01subunit may not be sufficient asa signal for degradations. Proteasomes and protein transport from to the cytosol are required for degradation

PII: 50962.8924(97)01020-9

the ER

Jeffrey L. Brodsky is in the Dept of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA; and Ardythe A. McCracken is in the Biology Dept, University of Nevada, Reno, NV 89557, USA. E-mails: jbrodsky+ @pitt.edu mccracke@cmb. unr.edu

151

focused on identifying an ER-specific protease. Otsu et al. showed that a cysteine protease from the ER of rat liver specifically binds to and degrades a heterologously expressed, misfolded form of lysozyme6. However, because this protease, known as ER-60, also degrades stable, resident ER proteins in vitro and has not been shown to hydrolyse other known substrates, its role, if any, in ER-associated protein degradation is not clear. No other ER-localized proteases that degrade specific ER proteins have been identified. Instead of finding an ER protease, evidence has accumulated that both soluble and integral ER membrane proteins are degraded by a cytosolic proteolytic machine7-15. This machine, known as the proteasome, degrades ubiquitinated and some non-ubiquitinated proteins and is essential for cell growth, division and immune system function3@. Proteasome-mediated protein degradation also requires a number of other factors that confer specificity and shepherd polypeptides to the degradation machinery3. A role for proteasomes in ER-associated protein degradation was suggested initially by the observations that they stud the cytosolic face of the ER membrane in secretory cells16 and that the yeast ER membrane contains a ubiquitin-conjugating enzyme”. However, the first clear indication that the proteasome degrades ER proteins came from studies on the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is an integral membrane protein that facilitates chloride transport across the apical plasma membrane of epithelial cells. The most common mutation in individuals with cystic fibrosis is a deletion of the phenylalanine at position 508 (AF508). Essentially all of the AF508 protein remains in the ER because an AT&dependent conformational change required for ER-to-Golgi transport is prevented18. Both immature wild-type and AF508 forms of CFTR retained in the ER are degraded rapidly in transfected cells, so those expressing only the AF508 species lack CFTR at the plasma membrane. To define the molecular mechanism through which CFTR is degraded in the ER, Jensen et al. used specific inhibitors to selectively prevent the ATP-dependent maturation of CFTR and its degradation in tissue culture cells’. The addition of lactacystin, a proteasome inhibitor, obstructed the degradation of the ER-retained forms of CFTR and increased the population of polyubiquitinated CFTR derivatives. Ward et al. then showed definitively that ubiquitination is required for proteasome-mediated degradation of CFTR by coexpression of a dominant-negative ubiquitin mutant, which impeded CFIR degradations. In addition, cells containing a temperature-sensitive ubiquitinactivating enzyme were unable to degrade CFTR at the nonpermissive temperature*. While it is clear from these studies that CFTR degradation is mediated by proteasomes, the trigger for CFTR maturation and the signal to ubiquitinate the immature forms remain obscure. However, CFTR associates with both cytosolic Hsc70~‘~ (70-kDa heatshock cognate proteins that bind and hydrolyse ATP) and with calnexinzo, a lumenal chaperone required for protein quality control in the ER. It is possible that this Hsc70 binding couples ATP hydrolysis to CFTR maturation, but the ATP-dependent step could also 152

involve phosphorylation of, or nucleotide binding by, CFTR. Only mature, transport-competent wildtype CFTR becomes freed from the chaperones1g,20, and, as described below, prolonged association with chaperones represents one mechanism by which aberrant proteins could be targeted for degradation. Studies on the major histocompatibility complex (MHC) class I molecule (MHCI) have also indicated that proteasomes degrade ER-associated proteins and have given further insights into how proteins may be transported out of the ER and thus presented to the proteasome. MHCI is an integral membrane protein that, in association with j32 microglobulin (j32m), presents virally derived peptides to cytolytic T cells. An MHCI-j32m-peptide complex assembles in the ER and then transits through the secretory pathway to the plasma membrane. To evade the immune system, mechanisms have evolved in human viruses to abolish MHCI function. In a study by Wiertz et aLg, the product of the human cytomegalovirus (CMV) US1 I gene, an ER membrane glycoprotein, was shown to target MHCI selectively to the cytosol where it was proteolysed. By contrast, j32m was secreted, demonstrating that US1 1 action is specific. Tissue culture cells expressing US1 1 grow as well as wild-type cells, also suggesting that US1 l-mediated protein degradation is not indiscriminate. As for the AF508 variant of CFTR (see above), MHCI degradation was inhibited by lactacysting. Another cytomegalovirus gene product, US2, also targets MHCI for rapid degradation. To investigate how US2 acts, Ploegh, Rapoport and their colleagues performed a series of coimmunoprecipitation experiments.‘O. Like US1 1, US2 targeted MHCI to the cytosol for degradation. When proteasome inhibitors were included in these reactions, stable complexes between MHCI molecules and proteasomes were detected. Amazingly, these experiments also detected an associL ation between MHCI and the SecGlp complex, the protein translocation channel in the ER membrane. If a reductant (dithiothreitol) was included so that unfolded MHCI polypeptides accumulated, complexes between the Sec6lp complex and MHCI were observed even in the absence of US2, suggesting that US2dependent and -independent retrotranslocation both occur via the Sec6lp complex. Together, these results indicate that ER export and import can use the same translocation machinery and identify one clear pathway by which proteins in the ER can be banished to the cytosol for degradation. ER-associated

protein

degradation

in yeast

How general is the phenomenon in which ERproteins are transported to and degradedby the cytosolic proteasome?To examine ER protein degradation in a genetically tractable organism in which factors required for proteolysismight be identified more readily, we developed an in vitro system using ER-derived microsomesand a solublemutated preprotein from the yeast Saccharomyces cerevisiae’l.Protein degradation required cytosol, hydrolysable ATP and was lessextensive in microsomesprepared from a strain lacking the lumenal chaperonecalnexin. This wasnot surprising sinceS.cerevisiae cellslacking calnexin mislocalize trends in CELL BIOLOGY

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1997

two ER-retained proteins to the plasma membrane21, suggesting that yeast calnexin, like its mammalian counterpart, is required for protein quality control. Because yeast ER-associated degradation was cytosol dependent, we examined whether the proteasome was required. In accordance with the studies described above, degradation was inhibited in vitro by lactacystin or when cytosol from a proteasome mutant strain was used, and was inhibited in vivo in strains containing mutations in the yeast proteasome12. Similarly, Hiller et al. discovered that the degradation of an ER-retained, misfolded form of the soluble hydrolase carboxypeptidase Y (CPY) was proteasome dependent and required the activity of a ubiquitin-conjugating enzyme13. In both studies, the soluble lumenal substrates targeted for degradation were observed in the cytosol, suggesting that they had been exported from the ER as a prelude to proteolysis12,13. It is not known whether the yeast Sec6lp complex is required for export, although the Wolf laboratory demonstrated that CPY ubiquitination and subsequent degradation requires the function of Derlp, an ER membrane protein of unknown function (see below)22. The degradation of ER membrane proteins in yeast, as in mammals, is also mediated by the proteasome. A temperature-sensitive mutation in the yeast gene encoding Sec6lp targets this protein and an associated subunit for ubiquitination and proteasome-mediated degradation when cells are grown at the nonpermissive temperature 14.Degradation of this mutant form of Sec6lp also requires two ubiquitin-conjugating enzymes. One of these, Ubc6p, is an ER membrane protein, and the other, Ubc7p, is a soluble protein whose localization has not yet been determined14. Ubc6p and Ubc7p are also required for ER-associated degradation of mutant CPY (Ref. 13). In addition, strains containing a mutation in the gene encoding the yeast p97/TRAP-2 protein, a component of the 26s proteasome, stabilize the rapidly degraded form of HMG-CoA reductase in yeast, and this defect can be rescued by heterologously expressing the human p97/TRAP-2 homologue’“. Substrate

selection

and regulated

degradation

For sometime, the dogma of protein biogenesishas been that nascent, secretedpolypeptides are translocated unidirectionally into the ERthrough an integral membrane channel or pore, but it is now apparent that this samepore, the SecGlp complex, also functions to extrude polypeptides from the ERIO.Although perhapssurprising, this is in fact consistent with previous observations suggestingthat polypeptides can oscillatebidirectionally in the channe123-25, hence the translocation channel hasno directional bias,and that glycopeptidescan be exported from yeast microsomes in vitro26. However, this lack of specificity within the channel itself meansthat selectionof proteinsfor degradation is likely to involve accessoryfactors in the ER lumen or membrane.Severalapproachesarecurrently being used to identify such components. One classof moleculesthat might mark these substratesfor export and degradationaremolecularchaperones, factors that facilitate protein folding and prevent aggregation. Protein degradation in bacteriaz7, trends in CELL BIOLOGY

(Vol.

7) April 1997

mitochondria2* and the yeast cytos01~~is known to require molecular chaperones.Chaperonesassociate with abnormally exposed regionsof proteins that are presentedduring protein folding, during protein translocation acrossbiological membranesand under denaturing conditions 30.Export from the ERand subsequent degradation may be achieved if a prolonged associationbetween lumenal chaperonesand a nonnative polypeptide occurs. Calnexin, a lumenal chaperone linked to ER-associated protein degradation”,“’ might be one such ‘sensory’ chaperone. Mammalian calnexin binds to the Z variant of human al-protease inhibitor (AlPiZ), a substratefor ER-associated protein degradation in both yeast32and mammalian cells33, and targets AlPiZ to the proteasome in mammalian microsomes31.As described above, calnexin also associateswith immature forms of CFTR in the ER before their degradatior?O.Another lumenal chaperone, BiP, is an Hsc70 that interacts with calnexin34 and displays kinetics and specificity of association with unfolded precursors that correlate with their degradation35,36. Theseobservations suggestthat the molecularchaperonesplay arole in targeting substrates for ER-associated protein degradation, but more direct evidence is clearly required to verify this hypothesis and to define their role. One ERmembraneprotein whoseregulated,specific degradation hasbeen well characterized is HMG-CoA reductase, an ER-resident enzyme that catalysesthe rate-limiting conversion of HMG-CoA to mevalonate during lipid biosynthesis. In yeast and mammalian cells, HMG-CoA reductaseis degradedin responseto increasedflux through the mevalonate pathway4. This proteolysis provides a rapid, irreversible and efficient meansto inhibit sterol and isoprenoid synthesiswhen molecules in the mevalonate pathway are plentiful. How HMG-CoA reductaseis specifically selectedfor degradation is unknown, but it may well involve interaction with a specific protein in the ERthat targets the native protein for degradation. A recently developed in vitro systemfrom mammalian cells3’and isolation of yeast mutants defective for a rapidly degradedHMG-CoA reductaseisozyme15should prove useful in elucidating this pathway and identifying the components involved. There are several examples of protein degradation in the ER of native proteins mediated by virally encoded geneproducts in addition to the CMV proteins describedabove (Ref. 38 and seeabove). One that has been particularly well characterized is the human immunodeficiency virus (HIV)-stimulated degradation of CD4, a receptor for HIV. An HIV gene product, VPU, interacts specifically with CD4 to facilitate its degradation3g.It will be interesting to learn whether VPU targets CD4 to the translocation machinery for delivery to the proteasomein a fashion analogousto degradation of MHCI facilitated by the CMV gene products US2 and USll. Characterization of factors such asthese will help in understanding viral pathogenesisand may well lead to the identification of cellular analogues. Taking a different approach to decipher the mechanismof ER-associated degradationand identify the componentsinvolved, Wolf13,22,Hamptonls, McCrackena, 153

Productive folding

BiP +-Productive folding

Aberrant folding

Calnexin

Aberrant folding A

Re-insertion of transmembrane helices into Sec61 p pore Activation Substrate retained in channel; chaperones bound

Channel is cleared; release of chaperones

(d)

(c)

Cytoplasm

Native ER membrane

Regulatory factor

Slippage of polypeptide into cytosol

protein

,II w

Lumen

Associated factor(s) induces conformational change or covalent modification in membrane protein

I

!

Association between cytosolic Hsc70 and exported polypeptide

omologue

J

Chaperone reassociation Cycles of ATP binding and hvdrolvsis facilitate unidirectional transport to the cytosol

c

FIGURE Models either

for soluble

and

folding

or conditions

that

resides

in the open

channel

misfolded through

suggested

previously’O.

of regulatory

p complex4’,

targeting may play

from

the ER to the cytosol. propose

posttranslational

for degradation. this

that

protein

and

for export of native

covalently

Retrotranslocation

a cytosolic

Hsc70

translocation

into

and

release

pore

activation

in the

may arise

ER membrane,

the conformation novel

into

through

the polypeptide ATP hydrolysis”‘,”

proteins

and

and

drives export and chaperones

(Ref.

protein,

association

between

resulting that

to the cytosol through either homologous

could

with

the

reductase4,

in the association are required

chaperones occurs

reticulum (ER) while aberrant the polypeptide

the chaperones be exported

and from

the

the

ER

pore, formed through the tetrameric the export of the polypeptide to the

of chaperones

molecular

the endoplasmic channel is cleared, 23-25. In this case,

domains

39) or HMC-CoA

in yeast

(d) Cytosolic

the pore

the ER lumen

ER. The translocation domains before

the association as CD4

of the native

signals’s,

into Prolonged

(b) Membrane-spanning

from such

ER membrane

to environmental

the ER lumen’.

polypeptide precursor.

inserted into the lipid bilayer of the for the reinsertion of membrane-spanning

p or Hrd3p,

with

of the nascent

(see text for details).

of the polypeptide interacts

ER. (a) Nascent polypeptides are translocated ‘, When folding proceeds, the translocation

to the translocating

degradation

or alter

responding

the

chaperones

bound

proteins

modify Hrdl

role upon

from delay

remain

after they have become would need to allow

degradation either

efflux molecular

of translocation

chaperones

The signal

(c)The

isozyme,

and

lumenal

in retrotranslocation

of pore’).

subsequent reductase

lumenal

channel

that

with

the completion

results

factors

selection

associate

while

of the Sec61 (‘Activation

action

inhibit

the translocation

association

protein

and

polypeptide

cytoplasm10

154

membrane

co- or posttranslationally

1

polypeptide, could

of molecular

for the degradation could

spontaneously

effect once

as has been

occur

through

chaperones

the and

of an HMC-CoA

the export the pore

of polypeptides is open23-2s.

We

a ratchet or pulling mechanism, as occurs during to Dna] (Ref. 30) may also be required for export.

Wends in CELL BIOLOGY

(Vol.

7) April 1997

and their colleagues, have used genetic screens to isolate degradation-defective mutants in yeast. DERI, a genecloned in the Wolf laboratory (seeabove)22,and HRDl and HRDS, isolatedby Hampton etaZ.r5,encode integral ERmembrane proteins of unknown function. It is tempting to speculate that these proteins may mark polypeptides for degradation, permit the degradation machinery to associatewith the ERmembrane or help escort membrane proteins to the cytosol for proteasome-mediateddegradation. A unifying model degradation?

for ER-associated

protein

play during the posttranslational translocation of nascentpolypeptides into the lumen of the ER’. Cytosolic DnaJhomologuesthat interact with Hsc70smight facilitate this processby anchoring the Hsc70sto the cytosolic face of the ER membrane and/or by stimulating ATP hydrolysis. Upon delivery to the cytosol, the polypeptide must be recognized by the proteasome. In somecases,the substrate is first tagged with ubiquitins,13,14*31, but in other casesdegradationisubiquitin independent10,12,37. Ubiquitin-independent degradation may occur becausethe polypeptides areeither unfolded or have specific chaperonesthat help feed substratesto the proteasome.The presenceof both ubiquitin-conjugating enzymesl’ and proteasomesl’jon the cytosolic face of the ER suggeststhat the degradation machinery can be tightly coupled to retrotranslocation.

As described above, it is now apparent that aberrant proteins and somenative proteins in the ERare selectedfor degradationand then exported to the cytosolfor degradation by the proteasome.In at leastone case,export is mediated by the Sec6lp ER translocation channel complex lo. While thesecharacteristics Concluding remarks of ER-associatedprotein degradation are clear, many An understanding of the processof ER-associated protein degradation has developed rapidly over the important questionsremained unanswered. Are molecular chaperonesrequired to select non-native pro- past year through disparate studies in yeast genetics teins for degradation? How are integral membrane and cell biology, immunology, and the pathology of proteins in the ERreturned to the translocation chan- human diseases.The resultsfrom these studiesallow us to speculateon a general mechanism, but several nel? Does the ER contain host proteins analogousto the viral geneproducts USZ,US11 or VPU that can taraspectsare still only poorly characterized. It remains get specific native proteins in the ERfor degradation? possible that ER-associateddegradation may utilize Is the Sec6lp complex required for the export of all ER both proteasome-dependentand -independent pathways, ashasbeen observedin one case3’.It is alsoforsubstratesdegradedby the proteasome?In an attempt to unify the existing data and to answer these ques- mally possible that the proteasome may indirectly tions, we present a model in Figure 1. activate another proteolytic machine that degrades In this model, we propose that, in the absenceof ERproteins. The ultimate goal will be to reconstitute proper folding, the chaperone complex remains as- ER-associated protein degradation biochemically sociated with the nascent polypeptide and that a with purified components. To this end, the analysis ‘decision’ is made by the translocation machinery to of yeast mutants and characterization of novel facre-export the polypeptide to the cytosol basedon the tors required for degradation has begun in earnest. kinetics of chaperone interaction. The nascent polyReferences peptide might remain tethered to Sec6lp while interacting with lumenal chaperones(Fig. la). Becausethe 1 BRODSKY, J. L. (1996) Trends Biochem. Sci. 21, 122-I 26 translocation pore probably remains open under 2 BONIFACINO, 1. S. and KLAUSNER, R. D. (1994) in Cellular these conditions23-25,soluble polypeptides may be Proteolytic Systems (Ciechanover, A. J. and Schwartz, A. L., eds), pp. 137-I 60, Wiley-Liss re-exported through the channel. In the caseof integral membrane proteins, the Sec6lp tetrameric 3 HOCHSTRASSER, M. (1995) Cm Opin. Cell Biol. 7, 215-223 complex41may partially dissociateto allow transmem4 HAMPTON, R., DIMSTER-DENK, D. and RINE, j. (1996) Trends brane domains to diffuse into the lipid bilayer. As Biochem. Sci. 21, 140-I 45 5 SHIN, I., LEE, 5. and STROMINGER, J. L. (1993) Science 259, suchdomainswould needtore-enter the pore for retrotranslocation, the pore must be gated or regulated to 1901-1904 6 OTSU, M., URADE, R., KITO, M., OMURA, F. and KIKUCHI, M. allow these segmentsto slip back into the channel (1995) 1, Biol. Cbem. 270,14958-l 4961 for re-export (Fig. lb). Native proteins in the lumen or lipid bilayer of the ERsuch asHMG-CoA reductase 7 JENSEN, T. J., LOO, M. A., PIND, S., WILLIAMS, D., may have partners in the ERmembrane that could be GOLDBERG, A. L. and RIORDAN, J. R. (1995) Cell 83,129-l 35 8 WARD, C. L., OMURA, S. and KOPITO, R. (1995) Cell83, induced to target the native protein for degradation, 121-127 perhapsby destabilizing its structure (Fig. l~)r~,~~.The destabilized polypeptides may then be recognized by 9 WIERTZ, E. J. H. J., JONES, T. R., SUN, L., BOCYO, M., ERchaperonesand targeted for degradation. CEUZE, H. 1. and PLOECH, H. L. (1996) Cell 84, 769-779 10 WEIRTZ, E. J. H. J. et ol. (1996) Nature 384,432-438 The energy required to drive protein export from the ER probably results from ATP hydrolysis. ATP is 11 MCCRACKEN, A. A. and BRODSKY, J. L. (1996) /. Cell Biol. 132, necessaryto export a glycopeptidez6 and polypeptide 291-298 substratestargetedfor degradationlO,llfrom the ERand 12 WERNER, E. D., BRODSKY, J. L. and MCCRACKEN, A. A. (1996) isrequiredto degradeother solubleERsubstrates33,4244. Proc. Nat/. Acod. Sci. U. 5. A. 93, 13797-I 3801 The most straightforward hypothesis would be that 13 HILLER, M. M., FINGER, A., SCHWEICER, M. and WOLF, D. H. (1996) Science 273, 1725-I 728 retrotranslocation is driven by ATP-requiring Hsc70s that help pull or ratchet polypeptides from the ER 14 BIEDERER, T., VOLKWEIN, C. and SOMMER, T. (1996) EMBO/. (Fig. Id), similar to the role that lumenal chaperones l&2069-2076 trends in CELL BIOLOGY

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7) April 1997

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15 16 17 18 Acknowledgements We thank Randy Hampton and Elizabeth Jones for their insightful discussions and comments on the manuscript. Work in the authors’ laboratories was supported by grants MCB-9506002 from the National Science Foundation, JFRA-602 from the American Cancer Society (to J. L. B.), and C789 from the Cystic Fibrosis Foundation and MCB-951042 from the National Science Foundation (to A. A. M.).

19 20 21 22 23 24 25 26 27 28 29

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30 31

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Memet, S.,Lilienbaum, A and Israel,A. (1997) Rapid isolation of mouse primary fibroblastsz a tool for the analysis of transgenic mice Technical Tips Online (http:l:wwv.elsevier.com~locate/tto) ‘101046 Sanzgiri, V.R.,Mangoli, S.H.,Ramchandani,J, and Mahajan. S.K. (1997) A simple method for studying ZacZfusions in la& Escberkbia coli hosts Technical T@sOn&e (http:/?vww,elsevier.com/locate.:’t TO1047 Vega-Palas.MA and Fed, R.J.(1997) A novel approach for analysis of chromatin using multiple restriction endonucleases Techwical Tips Online (http://www.elsevier.comilocateItto) TO1048 M. (1997) TRIzol” for plasmid DNA isolation Technical Tips Online (http://x+qv.elsevier.com:‘locate/tto) TO1050

Kniazeva.

Editor Adrian Bird, Institute for Cell and Molecular Biology at the University of Edinburgh

156

trends

in CELL BIOLOGY

(Vol.

7) April

1997