Mature A- and B-type lamins differ in the extent to which they interact with the nuclear membrane and thus represent an interesting model for studying the role of isoprenylation and carboxyl-methylation in membrane attachment. Both A- and B-type lamins are isoprenylated and carboxyl-methylated shortly after synthesis, but A-type lamins undergo a further proteolytic cleavage which results in the loss of the hydrophobically modified C terminus. Here, we have constructed mutants of chicken lamin A that differ in their abilities to serve as substrates for different post-translational processing events occurring at the C terminus of the wild-type precursor. In addition to studying full-length proteins, we have analyzed C-terminal end domains of lamin A, either alone or after fusion to reporter proteins. Mutant proteins were expressed in mammalian cells, and their membrane association was analyzed by immunofluorescence microscopy and subcellular fractionation. Our results provide information on the substrate specificity and subcellular localization of the lamin-A-specific protease. Moreover, they indicate that hydrophobic modifications of the C-terminal end domains account for the differential membrane-binding properties of A- and B-type lamins. Thus, some of the integral membrane proteins implicated in anchoring B-type lamins to the membrane may function as receptors for the isoprenylated and carboxyl-methylated C terminus.
REFERENCES
Aebi
U.
, Cohn
J.
, Buhle
L.
, Gerace
L.
(1986
). The nuclear lamina is a meshwork of intermediate-type filaments.
Nature
323
, 560
–564
Bailer
S. M.
, Eppenberger
H. M.
, Griffiths
G.
, Nigg
E. A.
(1991
). Characterization of a 54-kDa protein of the inner nuclear membrane: evidence for cell cycle dependent interaction with the nuclear lamina.
J. Cell Biol
114
, 389
–400
Beck
L. A.
, Hosick
T. J.
, Sinensky
M.
(1990
). Isoprenylation is required for the processing of the lamin A precursor.
J. Cell Biol
110
, 1489
–1499
Benavente
R.
, Krohne
G.
(1986
). Involvement of nuclear lamins in postmitotic reorganization of chromatin as demonstrated by microinjection of lamin antibodies.
J. Cell Biol
103
, 1847
–1854
Black
S. D.
(1992
). Development of hydrophobicity parameters for prenylated proteins.
Biochem. Biophys. Res. Commun
186
, 1437
–1442
Bossie
C. A.
, Sanders
M. M.
(1993
). A cDNA from Drosophila melanogaster encodes a lamin C-like intermediate filament protein.
J. Cell Sci
104
, 1263
–1272
Bridger
J. M.
, Kill
I. R.
, O'Farrell
M. O.
, Hutchison
C. J.
(1993
). Internal lamin structures within G1 nuclei of human dermal fibroblasts.
J. Cell Sci
104
, 297
–306
Burke
B.
, Gerace
L.
(1986
). A cell free system to study reassembly of the nuclear envelope at the end of mitosis.
Cell
44
, 639
–652
Butrynski
J. E.
, Jones
T. L. Z.
, Backlund
P. S.
Jr., Spiegel
A. M.
(1992
). Differential isoprenylation of carboxy-terminal mutants of an inhibitory G-protein-subunit: neither farnesylation nor geranylgeranylation is sufficient for membrane attachment.
Biochemistry
31
, 8030
–8035
Casey
P. J.
, Solski
P. A.
, Der
C. J.
, Buss
J. E.
(1989
). p21rasis modified by a farnesyl isoprenoid.
Proc. Nat. Acad. Sci. USA
26
, 8323
–8327
Casey
P. J.
(1992
). Biochemistry of protein prenylation.
J. Lipid Res
33
, 1731
–1740
Chavrier
P.
, Gorvel
J. P.
, Stelzer
E.
, Simons
K.
, Gruenberg
J.
, Zerial
M.
(1991
). Hypervariable C-terminal domain of rab proteins acts as a targeting signal.
Nature
353
, 769
–772
Chen
C.
, Okayama
H.
(1987
). High-efficiency transformation of mammalian cells by plasmid DNA.
Mol. Cell. Biol
7
, 2745
–2752
Clarke
S.
(1992
). Protein isoprenylation and methylation at carboxyl-terminal cysteine residues.
Annu. Rev. Biochem
61
, 355
–386
Cox
A. D.
, Der
C. J.
(1992
). Protein prenylation: more than just glue?.
Curr. Opin. Cell Biol
4
, 1008
–1016
Cox
A. D.
, Hisaka
M. M.
, Buss
J. E.
, Der
C. J.
(1992
). Specific isoprenoid modification is required for function of normal, but not oncogenic, ras protein.
Mol. Cell. Biol
12
, 2606
–2615
Dabauvalle
M.-C.
, Loos
K.
, Merkert
H.
, Scheer
U.
(1991
). Spontaneous assembly of pore complex-containing membranes (‘Annulate Lamellae’) in Xenopus egg extract in the absence of chromatin.
J. Cell Biol
112
, 1073
–1082
Dagenais
A.
, Bibor Hardy
V.
, Laliberte
J.-F.
, Royal
A.
, Simard
R.
(1985
). Detection in BHK cells of a precursor form for lamin A.
Exp. Cell Res
161
, 269
–276
Evan
G. I.
, Lewis
G. K.
, Ramsay
G.
, Bishop
J. M.
(1985
). Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product.
Mol. Cell. Biol
5
, 3610
–3616
Farnsworth
C. C.
, Wolda
S. L.
, Gelb
M. H.
, Glomset
J. A.
(1989
). Human lamin B contains a farnesylated cysteine residue.
J. Biol. Chem
264
, 20422
–20429
Fisher
D. Z.
, Chaudhary
N.
, Blobel
G.
(1986
). cDNA sequencing of nuclear lamins A and C reveals primary and secondary structural homology to intermediate filament proteins.
Proc. Nat. Acad. Sci. USA
83
, 6450
–6454
Foisner
R.
, Gerace
L.
(1993
). Integral membrane proteins of the nuclear envelope interact with lamins and chromosomes, and binding is modulated by mitotic phosphorylation.
Cell
73
, 1267
–1279
Gallant
P.
, Nigg
E. A.
(1992
). Cyclin B2 undergoes cell cycle-dependent nuclear translocation and, when expressed as a non-destructible mutant, causes mitotic arrest in HeLa cells.
J. Cell Biol
117
, 213
–224
Gerace
L.
, Blobel
G.
(1980
). The nuclear envelope lamina is reversibly depolymerized during mitosis.
Cell
19
, 277
–287
Gerace
L.
, Burke
B.
(1988
). Functional organization of the nuclear envelope.
Annu. Rev. Cell Biol
4
, 335
–374
Gieffers
C.
, Krohne
G.
(1991
). In vitro reconstitution of recombinant lamin A and a lamin A mutant lacking the carboxy-terminal tail.
Eur. J. Cell Biol
55
, 191
–199
Glass
J. R.
, Gerace
L.
(1990
). Lamins A and B bind and assemble at the surface of mitotic chromosomes.
J. Cell Biol
111
, 1047
–1057
Glomset
J. A.
, Gelb
M. H.
, Farnsworth
C. C.
(1990
). Prenyl proteins in eukaryotic cells: a new type of membrane anchor.
Trends Biochem. Sci
15
, 139
–142
Goldman
A. E.
, Moir
R. D.
, Montag-Lowy
M.
, Stewart
M.
, Goldman
R. D.
(1992
). Pathway of incorporation of microinjected lamin A into the nuclear envelope.
J. Cell Biol
119
, 725
–735
Goud
B.
(1992
). Small GTP-binding proteins as compartmental markers.
Semin. Cell Biol
3
, 301
–307
Hancock
J. F.
, Magee
A. I.
, Childs
J. E.
, Marshall
C. J.
(1989
). All ras proteins are polyisoprenylated but only some are palmitoylated.
Cell
57
, 1167
–1177
Hancock
J. F.
, Paterson
H.
, Marshall
C. J.
(1990
). A polybasic domain or palmitoylation is required in addition to the CAAX motif to localize p21rasto the plasma membrane.
Cell
63
, 133
–139
Hancock
J. F.
, Cadwallader
K.
, Paterson
H.
, Marshall
C. J.
(1991
). A CAAX or a CAAL motif and a second signal are sufficient for plasma membrane targeting of ras proteins.
EMBO J
10
, 4033
–4039
Heitlinger
E.
, Peter
M.
, Häner
M.
, Lustig
A.
, Aebi
U.
, Nigg
E. A.
(1991
). Expression of chicken lamin B2 in Escherichia coli: characterization of its structure, assembly, and molecular interactions.
J. Cell Biol
113
, 485
–495
Heitlinger
E.
, Peter
M.
, Lustig
A.
, Villiger
W.
, Nigg
E. A.
, Aebi
U.
(1992
). The role of the head and tail domain in lamin structure and assembly: analysis of bacterially expressed chicken lamin A and truncated B2 lamins.
J. Struct. Biol
108
, 74
–91
Höger
T. H.
, Zatloukal
K.
, Waizenegger
I.
, Krohne
G.
(1990
). Characterization of a second highly conserved B-type lamin present in cells previously thought to contain only a single B-type lamin.
Chromosoma
99
, 379
–390
Höger
T. H.
, Krohne
G.
, Kleinschmidt
J. A.
(1991
). Interaction of Xenopus lamins A and LII with chromatin in vitro mediated by a sequence element in the carboxyterminal domain.
Exp. Cell Res
197
, 280
–289
Holtz
D.
, Tanaka
R. A.
, Hartwig
J.
, McKeon
F.
(1989
). The CaaX motif of lamin A functions in conjunction with the nuclear localization signal to target assembly to the nuclear envelope.
Cell
59
, 969
–977
Horton
H.
, McMorrow
I.
, Burke
B.
(1992
). Independent expression and assembly properties of heterologous lamins A and C in murine embryonal carcinomas.
Eur. J. Cell Biol
57
, 172
–183
Inglese
J.
, Koch
W. J.
, Caron
M. G.
, Leftkowitz
R. J.
(1992
). Isoprenylation in regulation of signal transduction by G-protein-coupled receptor kinases.
Nature
359
, 147
–150
Kalderon
D.
, Roberts
B. L.
, Richardson
W. D.
, Smith
A. E.
(1984
). A short amino acid sequence able to specify nuclear location.
Cell
39
, 499
–509
Kinsella
B. T.
, Erdman
R. A.
, Maltese
W. A.
(1991
). Posttranslational modification of Ha-ras p21 by farnesyl versus geranylgeranyl isoprenoids is determined by the COOH-terminal amino acid.
Proc. Nat. Acad. Sci. USA
88
, 8934
–8938
Kitten
G. T.
, Nigg
E. A.
(1991
). The CaaX motif is required for isoprenylation, carboxyl methylation and nuclear membrane association of lamin B2.
J. Cell Biol
113
, 13
–23
Krek
W.
, Nigg
E. A.
(1989
). Structure and developmental expression of the chicken CDC2 kinase.
EMBO J
8
, 3071
–3078
Krek
W.
, Nigg
E. A.
(1991
). Mutations of p34cdc2phosphorylation sites induce premature mitotic events in HeLa cells: evidence for a double block to p34cdc2kinase activation in vertebrates.
EMBO J
10
, 3331
–3341
Krohne
G.
, Benavente
R.
(1986
). The nuclear lamins. A multigene family of proteins in evolution and differentiation.
Exp. Cell Res
162
, 1
–10
Krohne
G.
, Wolin
S. L.
, McKeon
F. D.
, Franke
W. W.
, Kirschner
M. W.
(1987
). Nuclear lamin LI of Xenopus laevis: cDNA cloning, amino acid sequence and binding specificity of a member of the lamin B subfamily.
EMBO J
6
, 3801
–3808
Krohne
G.
, Waizenegger
I.
, Höger
T. H.
(1989
). The conserved carboxy-terminal cysteine of nuclear lamins is essential for lamin association with the nuclear envelope.
J. Cell Biol
109
, 2003
–2011
Laliberte
J.-F.
, Dagenais
A.
, Filion
M.
, Bibor-Hardy
V.
, Simard
R.
, Royal
A.
(1984
). Identification of distinct messenger RNAs for nuclear lamin C and a putative precursor of nuclear lamin A.
J. Cell Biol
98
, 980
–985
Lebel
S.
, Lampron
C.
, Royal
A.
, Raymond
Y.
(1987
). Lamins A and C appear during retinoic acid-induced differentiation of mouse embryonal carcinoma cells.
J. Cell Biol
105
, 1099
–1104
Lehner
C. F.
, Furstenberger
G.
, Eppenberger
H. M.
, Nigg
E. A.
(1986
). Biogenesis of the nuclear lamina: In vivo synthesis and processing of nuclear protein precursors.
Proc. Nat. Acad. Sci. USA
83
, 2096
–2099
Lehner
C. F.
, Stick
R.
, Eppenberger
H. M.
, Nigg
E. A.
(1987
). Differential expression of nuclear lamin proteins during chicken development.
J. Cell Biol
105
, 577
–587
Loewinger
L.
, McKeon
F.
(1988
). Mutations in the nuclear lamin proteins resulting in their aberrant assembly in the cytoplasm.
EMBO J
7
, 2301
–2309
Lourim
D.
, Lin
J. J.-C.
(1989
). Expression of nuclear lamin A and muscle-specific proteins in differentiating muscle cells in ovo and in vitro.
J. Cell Biol
109
, 495
–504
Luderus
M.
, Graaf
A.
, Mattia
E.
, Blaauwen
J.
, Grande
M.
, de Jong
L.
, Driel
R.
(1992
). Binding of matrix attachment regions to lamin B1.
Cell
70
, 949
–959
Lutz
R. J.
, Trujillo
M. A.
, Denham
K. S.
, Wenger
L.
, Sinensky
M.
(1992
). Nucleoplasmic localization of prelamin A: implications for prenylation-dependent lamin A assembly into the nuclear lamina.
Proc. Nat. Acad. Sci. USA
89
, 3000
–3004
Maltese
W. A.
(1990
). Posttranslational modification of proteins by isoprenoids in mammalian cells.
FASEB J
4
, 3319
–3328
Marshall
C. J.
(1993
). Protein prenylation: a mediator of protein-protein interactions.
Science
259
, 1865
–1866
McKeon
F. D.
, Kirschner
M. W.
, Caput
D.
(1986
). Homologies in both primary and secondary structure between nuclear envelope and intermediate filament proteins.
Nature
319
, 463
–468
McKeon
F. D.
(1991
). Nuclear lamin proteins: domains required for nuclear targeting, assembly, and cell-cycle regulated dynamics.
Curr. Opin. Cell Biol
3
, 82
–86
Meier
J.
, Campbell
K. H. S.
, Ford
C. C.
, Stick
R.
, Hutchison
C. J.
(1991
). The role of lamin LIII in nuclear assembly and DNA replication, in cell-free extracts of Xenopus eggs.
J. Cell Sci
98
, 271
–279
Moores
S. L.
, Schaber
M. D.
, Mosser
S. D.
, Rands
E.
, O'Hara
M. B.
, Garsky
V. M.
, Marshall
M. S.
, Pompliano
D. L.
, Gibbs
J. B.
(1991
). Sequence dependence of protein isoprenylation.
J. Biol. Chem
266
, 14603
–14610
Munro
S.
, Pelham
H. R. B.
(1987
). A C-terminal signal prevents secretion of luminal ER proteins.
Cell
48
, 899
–907
Newport
J. W.
, Wilson
K. L.
, Dunphy
W. G.
(1990
). A lamin-independent pathway for nuclear envelope assembly.
J. Cell Biol
111
, 2247
–2259
Nigg
E. A.
(1989
). The nuclear envelope.
Curr. Opin. Cell. Biol
1
, 435
–440
Nigg
E. A.
(1992
). Assembly and cell cycle dynamics of the nuclear lamina.
Semin. Cell Biol
3
, 245
–253
Nigg
E. A.
, Kitten
G. T.
, Vorburger
K.
(1992
). Targeting lamin proteins to the nuclear envelope: the role of Caax box modifications.
Biochem. Soc. Trans
20
, 500
–504
Padan
R.
, Nainudel-Epszteyn
S.
, Goitein
R.
, Fainsod
A.
, Gruenbaum
Y.
(1990
). Isolation and characterization of the Drosophilia nuclear envelope otefin cDNA.
J. Biol. Chem
265
, 7808
–7813
Paddy
M. R.
, Agard
D. A.
, Sedat
J. W.
(1992
). An extended view of nuclear lamin structure, function, and dynamics.
Semin. Cell Biol
3
, 255
–266
Peter
M.
, Kitten
G. T.
, Lehner
C. F.
, Vorburger
K.
, Bailer
S. M.
, Maridor
G.
, Nigg
E. A.
(1989
). Cloning and sequencing of cDNA clones encoding chicken lamins A and B1 and comparison of the primary structures of vertebrate A-and B-type lamins.
J. Mol. Biol
208
, 393
–404
Peter
M.
, Nigg
E. A.
(1991
). Ectopic expression of an A-type lamin does not interfere with differentiation of lamin A-negative embryonal carcinoma cells.
J. Cell Sci
100
, 589
–598
Powell
L.
, Burke
B.
(1990
). Internuclear exchange of an inner nuclear membrane protein (p55) in heterokaryons: In vivo evidence for the interaction of p55 with the nuclear lamina.
J. Cell Biol
111
, 2225
–2234
Röber
R.-A.
, Weber
K.
, Osborn
M.
(1989
). Differential timing of nuclear lamin A/C expression in the various organs of the mouse embryo and the young animal: a developmental study.
Development
105
, 365
–378
Schafer
W. R.
, Rine
J.
(1992
). Protein prenylation- genes, enzymes, targets, and functions.
Annu. Rev. Genet
26
, 209
–.Schmidt-Zachmann
M. S.
, Nigg
E. A.
(1993
). Protein localization to the nucleolus: a search for targeting domains in nucleolin.
J. Cell Sci
105
, 799
–806
Seabra
M. C.
, Reiss
Y.
, Casey
P. J.
, Brown
M. S.
, Goldstein
J. L.
(1991
). Protein farnesyltransferase and geranylgeranyltransferase share a common alpha subunit.
Cell
65
, 429
–434
Senior
A.
, Gerace
L.
(1988
). Integral membrane proteins specific to the inner nuclear membrane and associated with the nuclear lamina.
J. Cell Biol
107
, 2029
–2036
Simos
G.
, Georgatos
S. D.
(1992
). The inner nuclear membrane protein p58 associates in vivo with a p58 kinase and the nuclear lamins.
EMBO J
11
, 4027
–4036
Stewart
C.
, Burke
B.
(1987
). Teratocarcinoma stem cells and early mouse embryos contain only a single major lamin polypeptide closely resembling lamin B.
Cell
51
, 383
–392
Stick
R.
, Angres
B.
, Lehner
C. F.
, Nigg
E. A.
(1988
). The fates ofchicken nuclear lamin proteins during mitosis: Evidence for a reversible redistribution of lamin B2 between inner nuclear membrane and elements of the endoplasmic reticulum.
J. Cell Biol
107
, 397
–406
Stick
R.
(1992
). The gene structure of Xenopus nuclear lamin A: a model for the evolution of A-type from B-type lamins by exon shuffling.
Chromosoma
101
, 566
–574
Ulitzur
N.
, Harel
A.
, Feinstein
N.
, Gruenbaum
Y.
(1992
). Lamin activity is essential for nuclear envelope assembly in a Drosophila embryo cell-free extract.
J. Cell Biol
119
, 17
–25
Vorburger
K.
, Kitten
G. T.
, Nigg
E. A.
(1989
). Modification of nuclear lamin proteins by a mevalonic acid derivative occurs in reticulocyte lysates and requires the cysteine residue of the C-terminal CXXM motif.
EMBO J
8
, 4007
–4013
Vorburger
K.
, Lehner
C. F.
, Kitten
G.
, Eppenberger
H. M.
, Nigg
E. A.
(1989
). A second higher vertebrate B-type lamin: cDNA sequence determination and in vitro processing of chicken lamin B2.
J. Mol. Biol
208
, 405
–415
Weber
K.
, Plessmann
U.
, Traub
P.
(1989
). Maturation of nuclear lamin A involves a specific carboxy-terminal trimming, which removes the polyisoprenylation site from the precursor; implications for the structure of the nuclear lamina.
FEBS Lett
257
, 411
–414
Wolin
S. L.
, Krohne
G.
, Kirschner
M. W.
(1987
). A new lamin in Xenopus somatic tissues displays strong homology to human lamin A.
EMBO J
6
, 3809
–3818
Worman
H. J.
, Yuan
J.
, Blobel
G.
, Georgatos
S. D.
(1988
). A lamin B receptor in the nuclar envelope.
Proc. Nat. Acad. Sci. USA
85
, 8531
–8534
Worman
H. J.
, Evans
C. D.
, Blobel
G.
(1990
). The lamin B receptor of the nuclear envelope inner membrane: A polytopic protein with eight potential transmembrane domains.
J. Cell Biol
111
, 1535
–1542
Yuan
J.
, Simos
G.
, Blobel
G.
, Georgatos
S. D.
(1991
). Binding of lamin A to polynucleosomes.
J. Biol. Chem
266
, 9211
–9215
This content is only available via PDF.
© 1994 by Company of Biologists
1994
-PolarityCFP.png?versionId=4839)
