Vol.

170,

July

31,

No.

2,

1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1990

Pages

A COMMON GOLGI

PEPTIDE

APPARATUS:

STRETCH

AMONG

STRUCTURAL

ENZYMES

SIMILARITY

LOCALIZED

OF

879-882

TO

THE

GOLGI-ASSOCIATED

GLYCOSYLTRANSFERASES Brad Bendiak The Biomembrane

Institute

and Department

of Pathobiology,

201 Elliott Avenue West, Seattle, Washington Received

.June 22,

University of Washington, 98119

1990

Summary: A common peptide motif has been discovered among a series of Golgi-localized glycosyltran.sferases. The peptide stretch, (Ser/Thr)-X-(Glu/Gln)-(ArgLys), always occurs near a hydrophobic domain close to the N-terminus of these enzymes which is believed to anchor them to the membrane lipid bilayer (Paulson and Colley, J. Biol. Chem., 264, 1761517618, 1989). The finding that this similar peptide motif is not associated with catalytic activity of these enzymes, and its presence near the hydrophobic domain suggest that the stretch may be involved in localization of these enzymes to the Golgi apparatus. Q1990 Academic

Pres:;,

Inc.

In eukaryotes, different

cellular membranes

proteins which enable each membrane not haphazardly intermingled,

are largely defined by characteristic

to carry out specific functions.

These proteins are

but come to be localized with high probability

to specific

organelles or suborganellar regions. A problem that remains unresolved is the mechanism by which many proteins come to reside in their membranes on a relatively long term basis, often referred to as “sorting” (1). As all proteins are initially translated from mRNA which can only define the linear sequence of a number compartmentalization

of amino acids, the information

for

of a particular protein to a specific organelle must reside, in essence,

in that protein’s sequence. The Golgi apparatus plays a central role in the traffic of integral membrane proteins through the cell (2). Even though many of these proteins are temporary residents in the Golgi apparatus en route to other destinations, there is a set of proteins which may be considered

permanent

residents of Golgi membranes,

and are known

to be marker

enzymes. Among these proteins are a number of the glycosyltransferases (3,4). The active sites of these enzymes are located in the lumen of the Golgi compartment, participate

in the assembly of the carbohydrate

where they

portions of glycoproteins and glycolipids. 0006-291x/90

879

$1.50

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

170,

No.

2,

1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

The structures of several Golgi-localized glycosyltransferases have been investigated; eight full-length sequences of five enzymes have been reported from different mammalian sources (5-13).

Extensive analysis of their sequences has now revealed a common stretch

which, it is postulated,

may be involved in localization

of these enzymes to the Golgi

apparatus. RESULTS AND DISCUSSION A

short

sequence

glycosyltransferases

reported

motif

was

discovered,

common

to

to date, which is shown in Fig. 1.

the

Golgi-localized

Although

exhaustive

computer searches have been performed by us (10) and others (5-9, 11-13) in the past, the common stretch was only found by consideration of the similar structures of Gln and Glu and the similar positive charge of Arg and Lys residues. The conserved stretch, (Ser/Thr)X-(Gln/Glu)-(Arg/Lys),

occurs adjacent to a nearby, more N-terminal

in four of the enzymes. Most important this common peptide hydrophobic domain.

for an understanding

Met-Pro sequence

of the possible function of

stretch is its position on the various glycosyltransferases

near a

In this regard, the overall domain structure of these enzymes is very

relevant, and Paulson and Colley (4) have presented a general structural hypothesis very similar to that shown in Fig. 2a. Essential common features of Golgi-localized

glycosyltransferases

are a catalytic

domain comprising much of the molecule toward the C-terminus, a “stem” region which is very susceptible to proteolytic cleavage and is probably solvent-exposed, and a hydrophobic domain near the N-terminus, which is probably embedded in the Golgi lipid bilayer. been postulated

(4) that the hydrophobic

It has

domain spans the membrane.

The common peptide stretch, in the enzymes examined to date, can occur either more toward the N-terminal but always occurs near it.

or C-terminal

of the protein than the hydrophobic

If this domain

actually

spans the lipid

domain,

bilayer

in all

glycosyltransferases, this would place the common peptide motif either in the cytoplasm, or in the Golgi lumen.

Met Met

Pro Pro

Lys Gly

1 Ile va1 GUY '=Y Ala Ala GUY

Murine 011-3 Galtransferase (5) Rat CY2-6 Sialyltransferase (6) Human 1X1-3 GalNActransferase "A" (7) "8" (8) Human al-3 Galtransferase Human, bovine 01-4 Galtransferase (9,101 Murine 01-4 Galtransferase (11,12) Bovine 011-3 Galtransferase (13)

Figure 1. Alignment of glycosyltransferase peptide motif. Numbers in brackets indicate references.

880

sequences having the common

structural

Vol.

170, No. 2, 1990

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Figure 2. Schematic representations of glycosyltransferaseswith the common structural motif (boxed) either C-terminal (a) or N-terminal (b) to the hydrophobic domain. It would be reasonable to surmise that if the common peptide retention

stretch were a

signal, that it should be located on the same side of the membrane

glycosyltransferases.

Such a situation could arise if both the N- and C-terminal

for all portions

of the proteins were located in the Golgi lumen, as shown in Fig. 2b. If this were the case, the hydrophobic

domain would comprise a hairpin loop, embedded in the membrane.

presence of hairpin precedented

loops in proteins

having one or more hydrophobic

The

domains

is

(14).

Also relevant is the fact that the similar peptide stretch occurs in enzymes which differ

in

their

catalytic

acetylgalactosamine,

properties

(one

transfers

one

transfers

N-terminal

Of the glycosyltransferases which have

sequences have established that proteolysis

of the initial

translation products occurs. This appears to be an artefact of purification. the polypeptide

N-

and three transfer galactose). Moreover, the common peptide stretch

is not associated in any way with catalytic activity. been purified,

sialic acid,

Cleavage of

occurs in the stem region, which releases the C-terminal

enzyme from the hydrophobic

domain.

portion of the

The freed C-terminal domain is a soluble enzyme

which retains full catalytic activity and substrate specificity. To date, N-terminal of the soluble enzyme forms (6,10,15-17) indicate that the tetrapeptide remain with the catalytic portion

of these enzymes after proteolysis

remains with the hydrophobic domain).

motif does not (but presumably

Therefore, the common peptide stretch cannot be

required for catalysis, and its location near the hydrophobic be involved in retention

sequences

domain suggests that it could

of these glycosyltansferases to the Golgi apparatus.

While it is also reasonable to postulate

many other functions for the common

peptide motif, its possible role as a Golgi retention signal cannot be ignored.

Of course,

other localization mechanisms may also exist, and this stretch may not be common to all proteins

resident

in the Golgi

apparatus,

or even to all glycosyltransferases.

881

The

Vol.

170,

No.

2,

1990

transferases reported

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

up to now synthesize the terminal structures of the carbohydrate

moieties of glycoproteins

and/or glycohpids; those Golgi-localized

enzymes which transfer

sugars nearer to the core of the carbohydrate units may be located in different cisternae of the Golgi stack (1). ACKNOWLEDGMENTS I thank Drs. S. Hakomori, comments in preparation

F. Yamamoto,

and S. Pawar for critical evaluation and helpful

of the manuscript.

Studies were supported by the Biomembrane

Institute. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Pfeffer, S.R. and Rothman, J.E. (1987) Ann. Rev. Biochem. 56, 829-852. Farquhar, M.G. and Palade, G.E. (1981) J. Cell Biol. 91, 77s-103s. MorrC, D.J., Merlin, L.M., and Keenan, T.W. (1969) Biochem. Biophys Rex Commun. 37, 813-819. Paulson, J.C. and Colley, K.J. (1989) J. Biol. Chem. 264, 1761517618. Larsen, R.D., Rajan, V.P., Ruff, M.M., Kukowska-Latallo, J., Cummings, R.D., and Lowe, J.B. (1989) Proc. Natl. Acad. Sci. lJ.SX 86, 8227-8231. Weinstein, J., Lee, E.U., McEntee, K., Lai, P.-H., and Paulson, J.C. (1987) J. Biol. Chem. 262, 17735-17743. Yamamoto, F., Marken, J., Tsuji, T., White, T., Clausen, H., and Hakomori, S. (1990) J. Biol. Chem. 265, 1146-1151. Yamamoto, F., Clausen, H., White, T., Marken, J., and Hakomori, S. (1990) Nature 345, 229-233. Masri, K.A., Appert, H.E., and Fukuda, M.N. (1988) Biochem. Biophys. Res. Commun. 157, 657-663. D’Agostaro, G., Bendiak, B., and Tropak, M. (1989) Eur. J. Biochem. 183, 211-217. Shaper, N.L., Hollis, G.F., Douglas, J.G., Kirsch, I.R., and Shaper, J.H. (1988) J. Biol. Chem. 263, 10420-10428. Nakazawa, K., Ando, T., Kimura, T., and Narimatsu, H. (1988) J. Biochem. (Tokyo) 104, 165-168. Joziasse, D.H., Shaper, J.H., Van den Eijnden, D.H., Van Tunen, A.J., and Shaper, N.L. (1989) J. Biol. Chem. 264, 14290-14297. Jennings, M.L. (1989) Ann. Rev. Biochem. 58, 999-1027. Appert, H.E., Rutherford, T.J., Tarr, G.E., Thomford, N.R., and McCorquodale, D.J. (1986) Biochem. Biophys. Res. Commun. 138, 224-229. Navaratnam, N., Ward, S., Fisher, C., Kuhn, N.J., Keen, J.N., and Findlay, J.B.C. (1988) Eur. J. Biochem. 171, 623-629. Clausen, H., White, T., Takio, K., Titani, K., Stroud, M., Holmes, E., Karkov, J., Thim, L., and Hakomori, S. (1990) J. Biol. Chem. 265, 1139-1145.

882

A common peptide stretch among enzymes localized to the Golgi apparatus: structural similarity of Golgi-associated glycosyltransferases.

A common peptide motif has been discovered among a series of Golgi-localized glycosyltransferases. The peptide stretch, (Ser/Thr)-X-(Glu/Gln)-(Arg/Lys...
267KB Sizes 0 Downloads 0 Views