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J. Biol. Chem., Vol. 280, Issue 36, 31890-31897, September 9, 2005

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Plasma Membrane Residence of Hyaluronan Synthase Is Coupled to Its Enzymatic Activity^*

Kirsi Rilla, Hanna Siiskonen, Andrew P. Spicer¶, Juha M. T. Hyttinen, Markku I. Tammi, and Raija H. Tammi

From the Department of Anatomy, University of Kuopio, FIN-70211 Kuopio, Finland and ^¶Texas A&M University System Health Science Center, Institute of Biosciences and Technology, Houston, Texas 77030

Hyaluronan is a multifunctional glycosaminoglycan up to 10⁷ Da molecular mass produced by the integral membrane glycosyltransferase, hyaluronan synthase (HAS). When expressed in keratinocytes, N-terminally tagged green fluorescent protein-HAS2 and -HAS3 isoenzymes were found to travel through endoplasmic reticulum (ER), Golgi, plasma membrane, and endocytic vesicles. A distinct enrichment of plasma membrane HAS was found in cell protrusions. The total turnover time of HAS3 was 4-5 h as judged by the green fluorescent protein signal decay and hyaluronan synthesis inhibition in cycloheximide-treated cells. The transfer from ER to Golgi took about 1 h, and the dwell time on the plasma membrane was less than 2 h in experiments with a relief and introduction, respectively, of brefeldin A. Constructs of HAS3 with 16- and 45-amino-acid C-terminal deletions mostly stayed within the ER, whereas a D216A missense mutant was localized within the Golgi complex but not the plasma membrane. Both types of mutations were almost or completely inactive, similar to the wild type enzyme that had its entry to the plasma membrane experimentally blocked by brefeldin A. Inhibition of hyaluronan synthesis by UDP-glucuronic acid starvation using 4-methyl-umbelliferone also prevented HAS access to the plasma membrane. The results demonstrate that 1) a latent pool of HAS exists within the ER-Golgi pathway; 2) this pool can be rapidly mobilized and activated by insertion into the plasma membrane; and 3) inhibition of HAS activity through mutation or substrate starvation results in exclusion of HAS from the plasma membrane.

Received for publication, April 29, 2005 , and in revised form, June 16, 2005.

^* This work was supported by Academy of Finland, Grants 54062 and 107173, and by grants from the North-Savo Cancer Fund, Kuopio University Foundation, Sigrid Juselius Foundation, Finnish Cancer Foundation, and by a research grant from Kuopio University Hospital. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains two supplemental videos.

To whom correspondence should be addressed: Dept. of Anatomy, University of Kuopio, P. O. B. 1627, FIN-70211 Kuopio, Finland. Tel.: 358-17-163010; Fax: 358-17-163032; E-mail: kirsi.rilla{at}uku.fi.

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