Tài liệu Báo cáo khoa học: Mammalian Gup1, a homolog of Saccharomyces cerevisiae glycerol

Mammalian Gup1, a homolog of Saccharomyces cerevisiae

glycerol uptake/transporter 1, acts as a negative regulator

for N-terminal palmitoylation of Sonic hedgehog

Yoichiro Abe1

, Yoshiko Kita1 and Takako Niikura1,2,*

1 Department of Pharmacology, Keio University School of Medicine, Tokyo, Japan

2 Department of Neurology, Georgetown University, Washington, DC, USA

Sonic hedgehog (Shh), a member of the vertebrate

Hedgehog (Hh) family [1–4], is an extracellular

secreted signaling molecule that is involved in embry￾onic patterning and organogenesis (for example, in the

dorsal–ventral polarity of the spinal cord and in the

anterior–posterior polarity in the limb bud) in a con￾centration-dependent manner [5].

Shh is initially translated as a precursor protein of

45 kDa. After excision of the signal sequence, it

undergoes automatic cleavage to release a biologically

Keywords

Gup1; hedgehog acyltransferase;

membrane-bound O-acyltransferase;

palmitoylation; Sonic hedgehog

Correspondence

Y. Abe, Department of Pharmacology,

Keio University School of Medicine,

35 Shinanomachi, Shinjuku-ku,

Tokyo 160-8582, Japan

Fax: +81 3 3359 8889

Tel: +81 3 5363 3750

E-mail: [email protected]

*Present address

Department of Neurology, Georgetown

University, Washington, DC, USA

(Received 21 August 2007, revised 9

November 2007, accepted 20 November

2007)

doi:10.1111/j.1742-4658.2007.06202.x

Mammalian glycerol uptake ⁄transporter 1 (Gup1), a homolog of Saccharo￾myces cerevisiae Gup1, is predicted to be a member of the membrane￾bound O-acyltransferase family and is highly homologous to mammalian

hedgehog acyltransferase, known as Skn, the homolog of the Drosoph￾ila skinny hedgehog gene product. Although mammalian Gup1 has a

sequence conserved among the membrane-bound O-acyltransferase family,

the histidine residue in the motif that is indispensable to the acyltransferase

activity of the family has been replaced with leucine. In this study, we

cloned Gup1 cDNA from adult mouse lung and examined whether Gup1

is involved in the regulation of N-terminal palmitoylation of Sonic hedge￾hog (Shh). Subcellular localization of mouse Gup1 was indistinguishable

from that of mouse Skn detected using the fluorescence of enhanced green

fluorescent protein that was fused to each C terminus of these proteins.

Gup1 and Skn were co-localized with an endoplasmic reticulum marker,

78 kDa glucose-regulated protein, suggesting that these two molecules

interact with overlapped targets, including Shh. In fact, full-length Shh

coprecipitated with FLAG-tagged Gup1 by immunoprecipitation using

anti-FLAG IgG. Ectopic expression of Gup1 with full-length Shh in cells

lacking endogenous Skn showed no hedgehog acyltransferase activity as

determined using the monoclonal antibody 5E1, which was found to recog￾nize the palmitoylated N-terminal signaling domain of Shh under denatur￾ing conditions. On the other hand, Gup1 interfered with the palmitoylation

of Shh catalyzed by endogenous Skn in COS7 and NSC34. These results

suggest that Gup1 is a negative regulator of N-terminal palmitoylation of

Shh and may contribute to the variety of biological actions of Shh.

Abbreviations

CHO, Chinese Hamster ovary; CM, conditioned medium; EGFP, enhanced green fluorescent protein; ER, endoplasmic reticulum; GRP78,

78-kDa glucose-regulated protein; Gup1, glycerol uptake ⁄ transporter 1; HHAT, hedgehog acyltransferase; HRP, horseradish peroxidase;

IP, immunoprecipitation; IRES, internal ribosome entry site; MBOAT, membrane-bound O-acyltransferase; Shh, sonic hedgehog; Shh-N,

N-terminal signaling domain of Shh without cholesterol modification; Shh-Np, autoprocessed N-terminal signaling domain of Shh;

TRITC, tetramethylrhodamine isothiocyanate.

318 FEBS Journal 275 (2008) 318–331 ª 2007 The Authors Journal compilation ª 2007 FEBS