The mechanisms by which the insulin-sensitive glucose transporter, GLUT4, is targeted and retained in a storage compartment near to the Golgi complex are poorly understood. Here we report that removal of the carboxyl-terminal acidic Pro(505)AspGluAsnAsp(509) sequence prevents the storage of GLUT4 in the VAMP-2 positive compartment adjacent to the Golgi complex (GSC), and results in its targeting to GLUT4-positive vesicles and Rab7-positive late endosomes. Storage of the truncated GLUT4 in the GSC is restored by substitution of Phe for the Tyr(502) residue adjacent to Pro(505) or by treatment of cells with the tyrosine kinase inhibitor genistein. Ablation of the Leu(489)Leu(490)-based motif prevents the targeting of GLUT4delta5 to GLUT4-positive-vesicles and late endosomes as well as the retention of GLUT4delta5Phe(502)by the GSC. These results are consisting with a model of GLUT4 transport in which the targeting of the protein from the TGN to the GSC is mediated by the Leu(489)Leu(490)-based motif and its release from the GSC involves Tyr(502)and the adjacent carboxyl-terminal Pro(505)AspGluAsnAsp(509) sequence.

REFERENCES

Akiyama
T.
,
Ishida
J.
,
Nakagawa
S.
,
Ogawara
H.
,
Watanabe
S.
,
Itoh
N.
,
Shibuya
M.
,
Fukami
Y.
(
1987
).
Genistein, a specific inhibitor of tyrosine-specific protein kinases.
J. Biol. Chem
262
,
5592
5595
Araki
S.
,
Yang
J.
,
Hashiramoto
M.
,
Tamori
Y.
,
Kasuga
M.
,
Holman
G. D.
(
1996
).
Subcellular trafficking kinetics of GLU4 mutated at the N-and C-terminal.
Biochem. J
315
,
153
159
Barriocanal
J.
,
Bonifacino
J. S.
,
Yuan
L.
,
Sandoval
I. V.
(
1986
).
Biosynthesis, glycosylation, movement through the Golgi system and transport to lysosomes by an N-linked carbohydrate independent mechanism of three lysosomal integral membrane proteins.
J. Biol. Chem
261
,
16755
16763
Bunn
C. R.
,
Jensaen
M. A.
,
Reed
B. C.
(
1999
).
Protein interactions with the glucose transporter binding protein GLUT1CBP that provide a link between GLUT1 and the cytoskeleton.
Mol. Biol. Cell
10
,
819
832
Cain
C. C.
,
Trimble
W. S.
,
Lienhard
G. E.
(
1992
).
Members of the VAMP family of synaptic vesicle proteins are components of glucose transporter-containing vesicles from rat adipocytes.
J. Biol. Chem
267
,
11681
11684
Camps
M.
,
Vilaro
S.
,
Testar
X.
,
Palacin
M.
,
Zorzano
A.
(
1994
).
High and polarized expression of GLUT1 glucose transporters in epithelial cells from mammary gland: acute down regulation of GLUT1 carriers by weaning.
Endocrinology
134
,
924
934
Chavrier
P.
,
Parton
R. G.
,
Hauri
H. P.
,
Simons
K.
,
Zerial
M.
(
1990
).
Localization of low molecular weight GTP binding proteins to exocytic and endocytic compartments.
Cell
62
,
317
329
Corvera
S.
,
Chawla
A.
,
Chakrabarty
R.
,
Joly
M.
,
Buxton
J.
,
Czech
M. P.
(
1994
).
A double leucine within the GLUT4 glucose transporter COOH-terminal domain functions as an endocytosis signal.
J. Cell Biol
126
,
979
989
Cosson
P.
,
Lefkir
Y.
,
Demolliere
C.
,
Letourneur
F.
(
1998
).
New COP1-binding motifs involved in ER retrieval.
EMBO J
17
,
6863
6870
Czech
M. P.
,
Buxton
J. M.
(
1993
).
Insulin action on the internalization of the GLUT4 glucose transporter in isolated rat adipocytes.
J. Biol. Chem
268
,
9187
9190
Czech
M. P.
,
Chawla
A.
,
Woon
C. W.
,
Buxton
J.
,
Armori
M.
,
Tang
W.
,
Joly
M.
,
Corvera
S.
(
1993
).
Exofacial epitope-tagged glucose transporter chimeras reveal COOH-terminal sequences governing cellular localization.
J. Cell Biol
123
,
127
135
Frost
S. C.
,
Lane
M. D.
(
1985
).
Evidence for the involvement of vicinal sulfhydryl groups in insulin-activated hexose transport by 3T3-L1 adipocytes.
J. Biol. Chem
260
,
2646
2652
Garippa
R. J.
,
Judge
T. W.
,
James
D. E.
,
Mc Graw
T. E.
) (
1994
).
The amino terminus of GLUT4 functions as an internalization motif but not an intracellular retention signal when substituted for the transferrin receptor cytoplasmic domain.
J. Cell Biol
124
,
705
715
Garippa
R. P.
,
Johnson
A.
,
Park
J.
,
Petrush
R. L.
,
McGraw
T. E.
(
1996
).
The carboxyll terminus of GLUT4 contains a serine-leucine-leucine sequence that functions as a potent internalization motif in chinese hamster ovary cells.
J. Biol. Chem
271
,
20660
20668
Griffiths
G.
,
Simons
K.
(
1986
).
The trans Golgi network: sorting at the exit site of the Golgi complex.
Science
234
,
438
443
Haney
P. M.
,
Slot
J. W.
,
Piper
R. C.
,
James
D. E.
,
Mueckler
M.
(
1991
).
Intracellular targeting of the insulin-regulatable glucose transporter (GLUT4) is isoform specific and independent of cell type.
J. Cell Biol
114
,
689
699
Haney
P. M.
,
Levy
M. A.
,
Strube
M. S.
,
Mueckler
M.
(
1995
).
Insulin-sensitive targeting of the GLUT4 glucose transporter in L6 myoblasts is conferred by its COOH-terminal cytoplasmic tail.
J. Cell Biol
129
,
641
658
Hauss
P.
,
Mazerolles
F.
,
Hivroz
C.
,
Lecomte
O.
,
Barbat
C.
,
Fischer
A.
(
1993
).
GF109203X, a specific PKC inhibitor, abrogates anti-CD3 antibody-induced upregulation of CD4+T cell adhesion to B cells.
Cell. Immunol
150
,
439
446
James
D. E.
,
Brown
R.
,
Navarro
J.
,
Pilch
P. F.
(
1988
).
Insulin regulatable tissues express a unique insulin-sensitive glucose transport protein.
Nature
333
,
183
185
Kunkel
T. A.
(
1985
).
The mutational specificity of DNA polymerase-beta during in vitro DNA synthesis. Production of frameshift, base substitution, and deletion mutations.
J. Biol. Chem
260
,
5787
5796
Laemmli
U. K.
(
1970
).
Cleveage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227
,
680
685
Letourneur
F.
,
Klausner
R. D.
(
1992
).
A novel di-leucine motif and a tyrosine-based motif independently mediate lysosomal targeting and endocytosis of CD3 chains.
Cell
69
,
1143
1157
Lopata
M. A.
,
Cleveland
D. W.
,
Sollner-Webb
B.
(
1984
).
High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethylsulfoxide or glycerol shock treatment.
Nucl. Acids Res
12
,
5707
5717
Macaulay
S. L.
,
Hewish
D. R.
,
Gough
K. H.
,
Stoichevska
V.
,
MacPherson
S. F.
,
Jagadish
S.
,
Ward
C. W.
(
1997
).
Functional studies in 3T3L1 cells support a role for SNARE proteins in insulin stimulation of GLUT4 translocation.
Biochem. J
324
,
217
224
Martin
S.
,
Tellam
J.
,
Livingstone
C.
,
Slot
J. W.
,
Gould
G. W.
,
James
D. E.
(
1996
).
The glucose transporter (GLUT4) and vesicle associated membrane protein (VAMP2) are segregated from recycling endosomes in insulin-sensitive cells.
J. Cell Biol
134
,
625
635
Martin
L. B.
,
Shewan
A.
,
Millar
C. A.
,
Gould
G. W.
,
James
D. E.
(
1998
).
Vesicle-associated membrane protein 2 plays a specific role in the insulin-dependent trafficking of the facilitative glucose transporter GLUT4 in 3T3-L1 adipocytes.
J. Biol. Chem
273
,
1444
1452
Miller
J.
,
Germain
R. N.
(
1986
).
Efficient cell surface expression of class II MHG molecules in the absence of associated invariant chain.
J. Exp. Med
164
,
1478
1489
Noda
T.
,
Farquhar
M. G.
(
1992
).
A non-autophagic pathway for diversion of ER secretory proteins to lysosomes.
J. Cell Biol
119
,
85
97
Oho
C.
,
Seino
S.
,
Takahashi
M.
(
1995
).
Expression and complex formation of soluble N-ethyl-maleimide-sensitive factor attachment protein (SNAP) receptors in clonal rat endocrine cells.
Neurosci. Lett
186
,
208
210
Olson
A. L.
,
Knight
J. B.
,
Pessin
J. E.
(
1997
).
Syntaxin 4, VAMP2, and/or VAMP3/cellubrevin are functional target membrane and vesicle SNAP receptors for insulin-stimulated GLUT4 translocation in adipocytes.
Mol. Cell. Biol
17
,
2425
2435
Piper
R. C.
,
Hess
L. J.
,
James
D. E.
(
1991
).
Differential sorting of two glucose transporters expressed in insulin-sensitive cells.
Am. J. Physiol
260
,
570
–.
Piper
R. C.
,
Tai
C.
,
Kulesza
P.
,
Pang
S.
,
Warnock
D.
,
Baenzinger
J.
,
Slot
J. W.
,
Geuze
H. J.
,
Puri
C.
,
James
D. E.
(
1993
).
GLUT-4 NH2terminus contains a phenylalanine-based targeting motif that regulates intracellular sequestration.
J. Cell Biol
121
,
1221
1232
Ralston
E.
,
Ploug
T.
(
1996
).
GLUT4 in cultured skeletal myotubes is segregated from the transferrin receptor and stored in vesicles associated with the TGN.
J. Cell Sci
109
,
2967
2978
Rapoport
I.
,
Chen
Y. G.
,
Cupers
P.
,
Shoelson
S. E.
,
Kirchhav-Sen
T.
(
1998
).
Dileucine-based sorting signals bind to the beta chain of AP-1 at a site distinct and regulated differently from the tyrosine-based motif-binding site.
EMBO J
17
,
2148
2155
Rodnick
K. J.
,
Slot
J. W.
,
Studelska
D. R.
,
Hanpeter
D. E.
,
Robinson
L. J.
,
Geuze
H. J.
,
James
D. E.
(
1992
).
Immunocytochemical andbiochemical studies of GLUT4 in rat skeletal muscle.
J. Biol. Chem
267
,
6278
6285
Sandoval
I. V.
,
Arredondo
J.
,
Alcalde
J.
,
Gonzalez-Noriega
A.
,
Vandekerckhove
J.
,
Jimenez
M. A.
,
Rico
M.
(
1994
).
The residues Leu(Ile)475-Ile(Leu, Val, Ala)476, contained in the extended cytoplasmic tail, are critical for targeting of the resident lysosomal integral membrane protein to lysosomes.
J. Biol. Chem
269
,
6622
6631
Saraste
J.
,
Palade
G. E.
,
Farquhar
M. G.
(
1986
).
Temperature-sensitive steps in the transport of secretory proteins through the Golgi complex in exocrine pancreatic cells.
Proc. Nat. Acad. Sci. USA
83
,
6425
6429
Slot
J. W.
,
Geuze
H. J.
,
Gigengack
S.
,
Lienhard
G. E.
,
James
D. E.
(
1991
).
Immunolocalization of the insulin regulatable glucose transporter in brown adipose tissue of the rat.
J. Cell Biol
113
,
123
135
Slot
J. W.
,
Geuze
H. J.
,
Gigengack
S.
,
James
D. E.
,
Lienhard
G. E.
(
1991
).
Translocation of the glucose transporter GLUT4 in cardiac myocites of the rat.
Proc. Nat. Acad. Sci. USA
88
,
7815
7819
Slot
J. W.
,
Garruti
G.
,
Martin
S.
,
Oorschot
V.
,
Posthuma
G.
,
Kraegen
E. W.
,
Laybutt
R.
,
Thilbaut
G.
,
James
D. E.
(
1997
).
Glucose transporter (GLUT-4) is targeted to secretory granules in rat atrial cardiomyocytes.
J. Cell Biol
137
,
1243
1254
Songyang
Z.
,
Cantley
L. C.
(
1995
).
Recognition and specificity in protein tyrosine kinase-mediated signalling.
Trends Biochem. Sci
20
,
470
475
Tamori
Y.
,
Hashiramoto
M.
,
Araki
S.
,
Kamata
Y.
,
Takahashi
M.
,
Kozaki
S.
,
Kasuga
M.
(
1996
).
Cleavage of vesicle-associated membrane protein (VAMP)-2 and cellubrevin on GLUT4-containing vesicles inhibits the translocation of GLUT4 in 3T3-L1 adipocytes.
Biochem. Biophys. Res. Commun
220
,
740
745
Traub
L. M.
,
Kornfeld
S.
(
1997
).
The trans-Golgi netrwork: a late secretory sorting station.
Curr. Opin. Cell Biol
9
,
527
533
Verhey
K. J.
,
Birnbaum
M. J.
(
1994
).
A Leu-Leu sequence is essential for COOH-terminal targeting signal of GLUT4 glucose transporter in fibroblasts.
J. Biol. Chem
269
,
2353
2356
Verhey
K. J.
,
Yeh
J. I.
,
Birnbaum
M. J.
(
1995
).
Distinct signals in the GLUT4 glucose transporter for internalization and for targeting to an insulin-responsive compartment.
J. Cell Biol
130
,
1071
1079
Yeh
J. I.
,
Verhey
K. J.
,
Birnbaum
M. J.
(
1995
).
Kinetical analysis of glucose transporter trafficking in fibroblasts and adipocytes.
Biochemistry
34
,
15523
15531
Yuan
L.
,
Barriocanal
J. G.
,
Bonifacino
J. S.
,
Sandoval
I. V.
(
1987
).
Two integral membrane proteins located in the cis-middle and trans-part of the Golgi system acquire sialylated N-linked carbohydrates and display different turnovers and sensitivity to cAMP-dependent phosphorylation.
J. Cell Biol
105
,
215
227
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