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The Journal of Experimental Biology 205, 1059-1067 (2002)
© 2002 The Company of Biologists Limited

Expression of Manduca sexta V-ATPase genes mvB, mvG and mvd is regulated by ecdysteroids

Stephan Reineke, Helmut Wieczorek and Hans Merzendorfer^*

Department of Biology/Chemistry, Division of Animal Physiology, University of Osnabrück, 49069 Osnabrück, Germany

^* Author for correspondence (e-mail: merzendorfer{at}biologie.uni-osnabrueck.de )

Accepted 7 February 2002

V-ATPases are complex proteins consisting of a peripheral, ATP-hydrolysing V₁ complex and a membrane-bound H⁺-translocating V_o complex. The plasma membrane V-ATPase from the tobacco hornworm (Manduca sexta) midgut is made up of eight different V₁ and four different V_o subunits. During starvation and moulting, V-ATPase activity decreases as a result of the dissociation of the V₁ complex from the V_o complex. To determine whether subunit biosynthesis is reduced during periods of enzyme inactivity, we measured the transcript levels and transcriptional activities of V-ATPase genes. Northern blots revealed the downregulation of almost all V-ATPase transcripts during starvation. During moulting, transcript levels of the three V-ATPase genes examined, mvB, mvG and mvd, also decreased, and this decrease was negatively correlated with the titre of 20-hydroxyecdysone (20-HE) and positively correlated with the titre of juvenile hormone (JH). To test the biological significance of these correlations, we injected both hormones into feeding larvae and measured transcript levels several hours later. A short-term increase and a long-term decrease in levels of mRNA were observed after 20-HE injection, whereas JH injection had no significant effect. Immunohistochemical studies of the midgut epithelium revealed that 20-HE injection led to changes in goblet cell morphology and in the subcellular distribution of the V₁ complex comparable with the situation during the moult and during starvation. Reporter gene assays in Sf21 cells using mvB, mvG and mvd promoters to initiate transcription of firefly luciferase led, after incubation of the cells with 20-HE, to results comparable with those obtained in the injection experiments. These findings suggest that putative ecdysone-responsive elements are present in all three promoters. Taken together, our results suggest that the expression of V-ATPase genes is controlled in a coordinated manner by ecdysteroids.

Key words: vacuolar H⁺-translocating ATPase, V-ATPase, Manduca sexta, promoter, transcription, ecdysteroid

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