On the variation of the major nuclear envelope (lamina) polypeptides

On the variation of the major nuclear envelope (lamina) polypeptides

Vol. April 93, No. BIOCHEMICAL 3, 1980 AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages aw-a72 14, 1980 On the Variation of the Major Nuclear E...

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Vol. April

93, No.

BIOCHEMICAL

3, 1980

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages aw-a72

14, 1980

On the Variation of the Major Nuclear Envelope (Lamina) Polypeptides Keith Ray Shelton, Valerie Hope Guthrie, and David Lee Cochran Department of Biochemistry and the MCV/VCUCancer Center, Virginia CommonwealthUniversity, RichmondVirginia 23298

Received

February

23,

1980

Three polypeptides which predominate in nuclear envelope and nuclear pore complex-lamina fractions are usually identified by their migration rates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Unfortunately, their migration relative to proteins frequently used as molecular weight standards can vary significantly under different electrophoresis conditions. However, the three maintain the sameorder of migration. They have been separated on the basis of both isoelectric point and apparent molecular weight and characterized by their tryptic peptides. Their identity as unique proteins has been established. One appears to be a cleavage product of another while the third has regions of unique sequence. This cleavage model is supported by an --in vitro cleavage reaction. Introduction Three major nuclear envelope polypeptides of the interphase nucleus (l-3).

These polypeptides predominate in the

nuclear pore complex-lamina, a fraction insoluble in nonionic detergents pared with rat liver titatively

are located in the periphery

(4,s) .

of the nuclear envelope which is In the avian erythrocyte,

or HeLa (cell nuclei,

a minor species (6).

the smaller of the three is quan-

However, this erythrocyte

appears to increase in amount under some-in vitro these proteins

polyacrylamide

identified

by their

gel electrophoresis

polypeptide

conditions

are hampered by the lack of specific

have been previously

as com-

(7).

Studies of

assays for them.

They

migration in sodium dodecyl sulfate-

(SDS-PAGE). This approach is inadequate

because reported molecular weights vary more widely than the differences tween the individual

polypeptides .

A more specific

achieved by two-dimensional electrophoresis, point separation followed by SDS-PAGE(8).

utilizing

identification first

be-

has been

isoelectric

In the present study, further 0006-291X/80/070867-06$01.00/0

867

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Copyright 0 1980 rights of reproduction

b.v Academrc Press, Inc. in anv form reserved.

Vol.

93, No.

3, 1980

BIOCHEMICAL

characterization peptides

has been achieved

purified

Materials

AND

BIOPHYSICAL

by obtaining

by two-dimensional

RESEARCH

tryptic

COMMUNICATIONS

peptide

maps of poly-

electrophoresis.

and Methods

Preparation of the chicken erythrocyte nuclear envelope fraction (detergent-washed) and separation of the polypeptides by isoelectric point separation and SDS-PAGE has been described (8). Coomassie blue R-stained spots were sectioned from gels which had been stored in 10% acetic acid. Radioiodination, tryptic cleavage, and application of the samples to cellulose thin layer plates was performed as described by Elder et -- al. (9). We and others have named the major nuclear envelope polypeptides on the basis of apparent molecular weight (1,2,7) but because of different determiFor this reason and because all nations, the designations vary widely. three polypeptides have their major concentration in the lamina region of the nucleus (see Introduction), we will refer to them by the recent designation lamin A, lamin B and lamin C (lo), replacing our P75, P71 and P68 designation.

Results

and Discussion

Variable

electrophoretic

lamin A vary dodecyl

electrophoresis

HeIa cell,

and chicken

system erythrocyte

SDS-PAGE dimension,

lamin B migrate mension,

between

a gradient

than serum albumin.

the relative other

weight

(14),

of 63 000 (unpublished

data).

in Figure

(13),

order

protein

it migrates

lamin A and

more rapidly

used here,

with

monomers between different order

different

gel system studies,

of SDS-PAGE migration, molecular

weight

values

(15).

where

to note that

respect

Thus, comparisons

they are identified

appear to be valid are obtained,

868

despite

lamin

an apparent

It is important

of lamin A and lamin B monomers with

in a single

1.

In the second di-

both migrate

of erythrocyte

An ex-

to each

did not change because homodimers of lamin A and lamin B reverse

migration

tive

buffers

In the second dimension,

migration

et -- al.

and serum albumin.

At the dilution

C is not detected. molecular

Iaemli

sodium

(6).

is presented

Shapiro

for

as lamin C from rat

identically

conditions

following

transferrin

gel with

migrate

weights

in a single

lamin A as well

ample of dependence on electrophoresis In the first

Reported molecular

from 80 000 (11) to 68 000 (12) although

sulfate

liver,

migration.

their

of the only by rela-

the fact

that

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76 67

35 Figure 1. Mobility of lamins A and B in different electrophoresis systems. Molecular weight standards were transferrin (76 000), bovine serum albumin (67 000), glutamate dehydrogenase (55 000) and glyceraldehyde 3-phosphate dehydrogenase (35 000). The erythrocyte lamina fraction was prepared as described under Materials and Methods. Electrophoresis in the first dimension (arrow) was performed in 5% acrylamide gels in the phosphate buffer system described by Shapiro et al. (13). This gel was applied to the top of a vertical S-20% acrylami~~l in the buffer system described by Laemli (14) and electrophoresis performed in the second dimension.

An in vitro

variation

chicken

erythrocyte

C under

some conditions.

2.

Panel

o is

the

in -relative

polypeptides,

there

An example

electropherogram

amounts

of the lamins.

can be an increased

of this

increase

With the

amount

is presented

of a predominantly

larnin

2c

of lamin in Fig.

A fraction

27

MWx103

Figure 2. Formation of a 68 OOO-dalton polypeptide iZiTZK&ed by extraction fram polyacrylamide gels in IO.10 M sodium phosphate (17) * The polypeptide, was boiled dodecyl sulfate:l.O% 2-mercaptoetiol, Treated samples were analyzed cated in each panel. in the Shapiro --et al. (13) buffers.

869

from lamin A. Lamin A as described previously (pH 3.5) :O.l% sodium for the time (min) indion 8.5% acrylamide gel

Vol.

93, No.

3, 1980

extracted

BIOCHEMICAL

from SDS-PAGE gels.

experiment,

this

fraction

in each panel.

peptide

in lamin C (68 000, C, as isolated

mational

change.

of the mild acid

0 and

There is,

27).

6 and 12).

panels

rate

oxidized as a result

In the

(minutes)

treatment

shown

is a signifi-

in a 61 OOO-dalton polya transient

This experiment

only the migration

presumably

the time

however,

might be a cleavage

COMMUNICATIONS

B and C are present.

effect

For instance,

form,

RESEARCH

at pH 3.5 for

from cells, is that

the reduced

of lamins

in lamin A (75 000) and an increase

(compare panels

explanation

Traces

BIOPHYSICAL

was boiled

The overall

cant decrease

AND

increase

indicates

product

that

of lamin A.

has varied

lamin

Another

because of a confor-

lamin A migrates of a different,

more rapidly stable

than

conformation

(16). The lamins gestion

that

respective

are unique polypeptides.

lamin C is a cleavage ionic

properties.

product

charge

approximately

point

isoelectric

lamin C could be a cleavage a two-dimensional compared (Fig. differ

gel, 3).

(panel

flect

incomplete

a) which

b)

did not contain cleavage.

is absent

being a cleavage C differing

with

and C in ionic with

product

a peptide

In contrast

a p1 approximately

occurring

0.7 pH units

A (8).

870

that from

spots

which

the vertical

and could

(under the + in

be from a fragment is consistent

lost with

with

as

peptides

any tryptic

of lamin A but is not consistent

as well,

tryptic

The area between

This evidence

numerous unique peptides. properties

test

in a second experiment

This could

to lamin C.

lamin A appears

lamins were obtained

few if

a).

Lamin A yields

from lamin C.

from their

To further

1251, and their

(panel

spots

only by conformation.

(panel c) yields

(8).

Lamin C (panel b) yields

lamin A is converted

species

radiolabeled

for the sug-

than lamin C and they both occur

the separate

from those found in lamin A

lines

erythrocyte,

variants

product,

support

of lamin A derives

In the chicken

to have only one more positive five

Additional

re-

panel

when lamin C

lamins A and

to lamins A and C, lamin B Lamin B differs

from lamins A

as a single

isoelectric

point

more acidic

than that

of lamin

Vol.

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BIOCHEMICAL

AND

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b

C

*

Ti=%

Radioiodinated tryptic peptides of the lamins. Samples, prepared as escrr ed under Materials and Methods, were spotted in the lower left corners. Chromatography (butanol:pyridine:acetic acid:water; 32.5:25:5:20), was performed in the first dimension. The dried plates were then moistened with electrophoresis buffer (acetic acid:formic acid:water, 15:5:80), and electrophoresis performed in the second dimension. Peptides were revealed by autoradiography. The detailed procedures have been described (9). The tryptic peptides of lamins A and C are in panels a and b while those of lamin B are in panel C. The chemical specific the

relationship

chemical

immunological

(quoted

in

(3)))

for

their

lamins.

cross-reaction

between

lamins

the very

similarity

evidence

of lamin

polypeptides. in

indicate

different

structures

common peptides

amino suggest

acid that

hand,

nuclear

A remains studies

.they are

the

a basis

A and C in

envelope

(16),

SDS-PAGE mapping

(15,16),

for

these

(1,

and the production proteins

(16).

and one-dimensional

cross-reactivity

related

and

The

and functions

(17)

for

and S. Ely

to be established.

imunological

compositions

permit

of lamins

by one-dimensional

B to lamin

other

A and C (1,

liver

topological

Gn the (3)),

and the rat

studies

They indicate

topographies

herein,

SDS-PAGE maps (16)

(quoted

(6)

similar

as detected

presented

These

of the

fraction

The relationship

lamins.

identification

for

the HeLa matrix

of the

S. Ely

of some

(17).

Acknowledgments This study Cancer Institute, lent secretarial

was supported by Grant CA 15923 awarded by the National Department of Health, Education and Welfare. The excelskill of Judy L. Watts is appreciated.

References 1.

Gerace,

2.

E~Y, S.,

L. , Blum, D’Arcy,

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BIOCHEMICAL

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Elder, J.H., Pickett, R.A., Biol. -Chem. 252, 6510-6515.

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