All intermediate filament proteins consist of an alpha-helical rod domain flanked by non-helical N-terminal head and C-terminal tail domains. The roles of the non-helical domains of various intermediate filament proteins in the assembly and co-assembly of higher-order filamentous structures have been studied by many groups but with quite contradictory results. Type III intermediate filaments are unique in that they can form homopolymers both in vitro and in vivo. The expression and assembly characteristics of carboxy- and amino-terminal deletion mutants of glial fibrillary acidic protein (GFAP), an astrocyte-specific type III intermediate filament protein, were examined by transient transfections of either vimentin-positive or vimentin-negative variants of human adrenocarcinoma-derived SW13 cell lines. Whereas complete deletion of the C-terminal tail domain of GFAP results in the formation of polymorphic aggregates, both intranuclear and cytoplasmic in self-assembly experiments, efficient co-assembly of these tail-less GFAP mutants with vimentin can be achieved as long as the KLLEGEE sequence at the C-terminal end of the rod domain is preserved. Up to one-fifth of the C-terminal end of the tail domain can be deleted without affecting the capability of GFAP to self-assemble. The highly conserved RDG-containing motif in the tail domain may be important for self-assembly but is not sufficient. The entire head domain seems to be required for self-assembly. All N-terminal deletion mutants of GFAP share the same phenotype of diffuse cytoplasmic staining when expressed in vimentin-negative SW13 cells. Although co-assembly with vimentin can still be achieved with completely head-less GFAP, preservation of some of the head domain greatly enhanced the efficiency. Our results form the basis for further, more detailed mapping of the essential regions in filament assembly of GFAP and other type III IFs.

REFERENCES

Albers
K.
,
Fuchs
E.
(
1987
).
The expression of mutant epidermal keratin cDNAs transfected in simple epithelial and squamous cell carcinoma lines.
J. Cell Biol
105
,
791
806
Albers
K.
,
Fuchs
E.
(
1989
).
Expression of mutant keratin cDNAs in epithelial cells reveals possible mechanisms for initiation and assembly of intermediate filaments.
J. Cell Biol
108
,
1477
1493
Bader
B. L.
,
Magin
T. M.
,
Hatzfeld
M.
,
Franke
W. W.
(
1986
).
Amino acid sequence and gene organization of cytokeratin no. 19, an exceptional tail-less intermediate filament protein.
EMBOJ
5
,
1865
1875
Bader
B. L.
,
Magin
T. M.
,
Freudenmann
M.
,
Stumpp
S.
,
Franke
W. W.
(
1991
).
Intermediate filaments formed de novo from tail-less cytokeratins in the cytoplasm and in the nucleus.
J. Cell Biol
115
,
1293
1307
Birkenberger
L.
,
Ip
W.
(
1990
).
Properties of the desmin tail domain: Studies using synthetic peptide antibodies.
J. Cell Biol
111
,
2063
2075
Bloemendal
H.
,
Pieper
F. R.
(
1989
).
Intermediate filaments: known structure, unknown function.
Biochim. Biophys. Acta
1007
,
245
253
Chin
S. S. M.
,
Liem
R. K. H.
(
1989
).
Expression of rat neurofilament proteins NF-L and NF-M in transfected non-neuronal cells.
Eur. J. Cell Biol
50
,
475
490
Chin
S. S. M.
,
Macioce
P.
,
Liem
R. K. H.
(
1991
).
Effects of truncated neurofilament proteins on the endogenous intermediate filaments in transfected fibroblasts.
J. Cell Sci
99
,
335
350
Ching
G.-Y.
,
Liem
R. K. H.
(
1993
).
Assembly of type IV neuronal intermediate filaments in non neuronal cells in the absence of preexisting cytoplasmic intermediate filaments.
J. Cell Biol
122
,
1323
1335
Conway
J. F.
,
Parry
D. A. D.
(
1988
).
intermediate filament structure: 3. Analysis of sequence homologies.
Int. J. Biol. Macromol
10
,
79
98
Crewther
W. G.
,
Dowling
L. M.
,
Steinert
P. M.
,
Parry
D. A. D.
(
1983
).
Structure of intermediate filaments.
Int. J. Biol. Macromol
5
,
267
274
Eckelt
A.
,
Herrmann
H.
,
Franke
W. W.
(
1992
).
Assembly of a tail-less mutant of the intermediate filament protein, vimentin, in vitro and in vivo.
Eur. J. Cell Biol
58
,
319
330
Franke
W.
(
1987
).
Homology of a conserved sequence in the tail domain of intermediate filament proteins with the loop region of calcium binding proteins.
Cell Biol. Int. Rep
11
,
831
–.
Geisler
N.
,
Weber
K.
(
1982
).
The amino acid sequence of chicken muscle desmin provides a common structural model for intermediate filament proteins.
EMBO. J
1
,
1649
1656
Geisler
N.
,
Weber
K.
(
1983
).
Amino acid sequence data on glial fibrillary acidic protein (GFA): implications for the subdivision of intermediate filaments into epithelial and non-epithelial members.
EMBOJ
2
,
2059
2063
Gill
S. R.
,
Wong
P. C.
,
Monteiro
M. J.
,
Cleveland
D. W.
(
1990
).
Assembly properties of dominant and recessive mutations in the small mouse neurofilament (NF-L) subunit.
J. Cell Biol
111
,
2005
2019
Graham
F. L.
,
van der Eb
A. J.
(
1973
).
A new technique for the assay of infectivity of human adenovirus 5 DNA.
Virology
52
,
456
467
Hatzfeld
M.
,
Weber
K.
(
1990
).
Tailless keratins assemble into regular intermediate filaments in vitro.
J. Cell Sci
97
,
317
324
Hatzfeld
M.
,
Weber
K.
(
1991
).
Modulation of keratin intermediate filament assembly by single amino acid exchanges in the consensus sequence at the C-terminal end of the rod domain.
J. Cell Sci
99
,
351
362
Herrmann
H.
,
Hofmann
I.
,
Franke
W. W.
(
1992
).
Identification of a nonapeptide motif in the vimentin head domain involved in intermediate filament assembly.
J. Mol. Biol
223
,
637
650
Kaufmann
E.
,
Weber
K.
,
Geisler
N.
(
1985
).
Intermediate filament forming ability of desmin derivatives lacking either the amino-terminal 67 or the carboxy-terminal 27 residues.
J. Mol. Biol
185
,
733
742
Kouklis
P. D.
,
Papamarcaki
T.
,
Merdes
A.
,
Georgatos
S. D.
(
1991
).
A potential role for the COOH-terminal domain in the lateral packing of type III intermediate filaments.
J. Cell Biol
114
,
773
786
Krauss
S.
,
Franke
W.
(
1990
).
Organization and sequence of the human gene encoding cytokeratin 8.
Gene
86
,
241
249
Lee
M. K.
,
Xu
Z.
,
Wong
P. C.
,
Cleveland
D. W.
(
1993
).
Neurofilaments are obligate heteropolymers in vivo.
J. Cell Biol
122
,
1337
1350
Leonard
D. G. B.
,
Gorham
J. D.
,
Cole
P.
,
Greene
L. A.
,
Ziff
E. B.
(
1988
).
A nerve growth factor-regulated messenger RNA encodes a new intermediate filament protein.
J. Cell Biol
106
,
181
193
Lu
X.
,
Lane
E. B.
(
1990
).
Retrovirus-mediated transgenic keratin expression in cultured fibroblasts: Specific domain functions in keratin stablization and filament formation.
Cell
62
,
681
696
McCormick
M. B.
,
Kouklis
P.
,
Syder
A.
,
Fuchs
E.
(
1993
).
The roles of thd rod end and the tail in vimentin IF assembly and IF network formation.
J. Cell Biol
122
,
395
407
McKeon
F.
(
1991
).
Nuclear lamin proteins: domains required for nuclear targeting, assembly and cell-cycle-regulated dynamics.
Curr. Opin. Cell Biol
3
,
82
86
McLachlax
A. D.
,
Stewart
M.
(
1982
).
Periodic charge distribution in the intermediate filament proteins desmin and vimentin.
J. Mol. Biol
162
,
693
698
Nakamura
Y.
,
Takeda
M.
,
Aimoto
S.
,
Nishimura
T.
(
1992
).
Assembly regulatory domain of glial fibrillary acidic protein.
J. Biol. Chem
267
,
23269
23274
O'Shea
E. K.
,
Klemm
J. D.
,
Kim
P. S.
,
Albert
T.
(
1991
).
X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled-coil.
Science
254
,
539
544
Parker
G. A.
,
Stark
G. R.
(
1979
).
Regulation of simian virus 40 transcription: Sensitive analysis of the RNA species present early in infections by virus or viral DNA.
Virology
31
,
360
369
Parry
D. A. D.
,
Steinert
P. M.
(
1992
).
Intermediate filament structure.
Curr. Opin. Cell Biol
4
,
94
98
Quinlan
R. A.
,
Moir
R. D.
,
Stewart
M.
(
1989
).
Expression in Escherichia coli of fragments of glial fibrillary acidic protein: characterization, assembly properties and paracrystal formation.
J. Cell Sci
93
,
71
83
Raats
J. M. H.
,
Pieper
F. R.
,
Vree Egberts
W. T. M.
,
Verrijp
K. N.
,
Ramaekers
F. C. S.
,
Bloemendal
H.
(
1990
).
Assembly of amino-terminally deleted desmin in vimentin-free cells.
J. Cell Biol
111
,
1971
1985
Raats
J. M. H.
,
Henderik
J. B. J.
,
Verdijk
M.
,
Bloemendal
H.
(
1991
).
Assembly of carboxy-terminally deleted desmin in vimentin-free cells.
Eur. J. Cell Biol
56
,
84
103
Raats
J. M.
,
Gerards
W. L. H.
,
Schreuder
M. I.
,
Bloemendal
H.
(
1992
).
Biochemical and structural aspects of transiently and stably expressed mutant desmin in vimentin-free and vimentin-containing cells.
Eur. J. Cell Biol
58
,
108
127
Reeves
S.
,
Helman
L. J.
,
Allison
A.
,
Israel
M. A.
(
1989
).
Molecular cloning and primary structure of human glial fibrillary acidic protein.
Proc. Nat. Acad. Sci. USA
86
,
5178
5182
Sanger
F.
,
Coulsen
A. R.
,
Barrel
B. G.
,
Smith
J. H.
,
Roe
B.
(
1980
).
Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing.
J. Mol. Biol
143
,
161
178
Sarria
A. J.
,
Nordeen
S. K.
,
Evans
R. M.
(
1990
).
Regulated expression of vimentin cDNA in cells in the presence and absence of a preexisting vimentin filament network.
J. Cell Biol
111
,
553
565
Silver
P. A.
(
1991
).
How proteins enter the nucleus.
Cell
64
,
489
497
Stasiak
P. C.
,
Purkis
P. E.
,
Leigh
I. M.
,
Lane
E. B.
(
1989
).
Keratin 19: predicted amino acid sequence and broad tissue distribution suggest it evolved from keratinocyte keratin.
J. Invest. Derm
92
,
707
716
Steinert
P. M.
,
Steven
A. C.
,
Roop
D. R.
(
1985
).
The molecular biology of intermediate filaments.
Cell
42
,
411
420
Steinert
P. M.
,
Roop
D. R.
(
1988
).
Molecular and cellular biology of intermediate filaments.
Annu. Rev. Biochem
57
,
593
625
Steinert
P. M.
,
Parry
D. A. D.
(
1993
).
The conserved H1 domain of the type II keratin 1 chain plays an essential role in the alignment of nearest neighbor molecules in mouse and human keratin 1/keratin 10 intermediate filaments at the two-to four-molecule level of structure.
J. Biol. Chem
268
,
2878
2887
Traub
P.
,
Vorgias
C.
(
1983
).
Involvement of the N-terminal peptide of vimentin in the formation of intermediate filaments.
J. Cell Sci
63
,
43
67
van den Heuvel
R. M. M.
,
van Eys
G. J. J. M.
,
Ramaekers
F. C. S.
,
Quaz
W. J.
,
Vree Egberts
W. T. M.
,
Schaart
G.
,
Cuypers
H. T. M.
(
1987
).
Intermediate filament formation after transfection with modified hamster vimentin and desmin genes.
J. Cell Sci
88
,
475
482
Wang
E.
,
Cairncross
J. G.
,
Liem
R. K. H.
(
1984
).
Identification of glial filament protein and vimentin in the same intermediate filament system in human glioma cells.
Proc. Nat. Acad. Sci. USA
81
,
2102
2106
Westermark
B.
(
1973
).
The deficient density-dependent growth control of human malignant glioma cells and virus-transformed glia-like cells in culture.
Int. J. Cancer
12
,
438
451
Wong
P. C.
,
Cleveland
D. W.
(
1990
).
Characterization of domain and recessive assembly-defective mutations in mouse neurofilament NF-M.
J. Cell Biol
111
,
1987
2003
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