JETLAG resets the Drosophila circadian clock by promoting light-induced degradation of TIMELESS

Abstract

Organisms ranging from bacteria to humans synchronize their internal clocks to daily cycles of light and dark. Photic entrainment of the Drosophila clock is mediated by proteasomal degradation of the clock protein TIMELESS (TIM). We have identified mutations in jetlag-a gene coding for an F-box protein with leucine-rich repeats-that result in reduced light sensitivity of the circadian clock. Mutant flies show rhythmic behavior in constant light, reduced phase shifts in response to light pulses, and reduced light-dependent degradation of TIM. Expression of JET along with the circadian photoreceptor cryptochrome (CRY) in cultured S2R cells confers light-dependent degradation onto TIM, thereby reconstituting the acute response + of the circadian clock to light in a cell culture system. Our results suggest that JET is essential for resetting the clock by transmitting light signals from CRY to TIM.

Fig. 1

Mutant phenotypes and mapping of the jet mutations. (A) Activity records of representative wild-type (y w) and mutant flies in LL and DD. The gray and black bars at the top indicate the LD cycle. (B) Activity records showing average activity of wild-type and mutant flies (n = 13 and 15, respectively) in a “jet lag” experiment. The flies were transferred from one LD schedule (indicated by the bars at the top) to another with an 8-hour delay (indicated by the bars at the bottom) during the third day. Red dots indicate evening onsets, and the blue arrowhead indicates a startle response at lights-off after the transfer to a new LD schedule. (C) Schematic representation of the JET protein. Arrows indicate the Phe → Ile (c)and Ser → Leu (r) substitutions in the fourth and fifth LRRs, respectively. (D) Alignment of the fourth and fifth LRRs of insect, mouse, and human F-box proteins with highest similarity to JET. See supporting online material for GenBank accession numbers. (E) Normal circadian expression of per in peripheral clocks of jet^c mutants. Flies (21 control, 22 mutant) carrying a per-luciferase (BG-luc) transgene were entrained to a 12 hour:12 hour LD cycle and were individually monitored in DD for ~5 days; data are mean numbers of counts per second.

Fig. 2

Reduced responses to light pulses in jet mutant flies. (A) Behavioral response to phase-delaying and phase-advancing light pulses. All differences between control and mutant flies for both alleles and both zeitgeber times (ZTs) were significant [P < 0.0001 by analysis of variance (ANOVA)]. In this and subsequent figures, error bars denote SEM. (B) Rescue of reduced light sensitivity of jet mutants with a UAS-jet transgene. Phase delays in response to light pulses at ZT 16 in jet^c flies with a UAS-jet transgene and either a cry- or tim-Gal4 driver were assayed as in (A). Two independent UAS-jet lines, A and B, produced similar results. ^xP = 0.06, *P < 0.01, **P < 0.001 by ANOVA. (C) Reduced light-dependent degradation of TIM in clock neurons of jet^c mutants. Representative TIM staining in small ventral lateral neurons is shown 1 hour after a 2-min light pulse (LP) at ZT 16. TIM staining without LP is shown for comparison; 8 to 10 fly brains were examined per condition. (D) Rescue of the TIM response to light in clock neurons of jet^c mutants with a UAS-jet transgene. jet^c mutants with a tim-Gal4 driver alone or with a UAS-jet transgene and a tim-Gal4 driver were assayed as in (C).

Fig. 3

Light-dependent degradation of TIM in S2R+ cells. (A) Light-induced degradation of TIM is mediated by CRY, TIM, and the proteasome pathway. S2R+ cells were transiently transfected with pAc-tim along with the pIZ vector or FLAG epitope-tagged pIZ-jet (pIZ-FLAG-jet), with or without MYC epitope-tagged pIZ-cry (pIZ-MYC-cry). MYC-CRY and FLAG-JET protein levels were determined with antibodies to MYC and JET, respectively. (B) JET does not promote degradation of PER. Hemagglutinin (HA)-tagged pAct-per and pIZ-MYC-cry were transfected with or without pIZ-FLAG-jet.(C) Mutant JET proteins are less effective at promoting TIM degradation than wild-type JET, and the r version of mutant JET is less stable than wild-type JET. Cells were transfected with pIZ or pIZ-FLAG-jet (wild-type or mutant) at one-fifth the concentration used in (A) along with pIZ-tim and pIZ-MYC-cry.(D) JET promotes light-dependent ubiquitination of TIM. S2R+ cells were transfected with HA-tagged ubiquitin (UB) under the control of a heat shock promoter along with pAc-tim and wild-type or mutant pIZ-FLAG-jet, with or without pIZ-MYC-cry. Transfected cells were kept at 25°C. Cell extracts were immunoprecipitated with antibody to TIM, and the relative amounts of ubiquitinated TIM were measured with antibodies to HA and TIM. (E) JET interacts with SkpA in vitro. Purified JET protein fused to maltose binding protein (MBP) was incubated with either glutathione S-transferase (GST) or GST-SkpA fusion protein. MBP-JET was detected with an antibody to JET (top), and GST and GST-SkpA were visualized by Coomassie staining (bottom). (F) JET physically associates with TIM in S2R+ cells in a light-dependent manner. Extracts from cells transfected with pAc-tim, pIZ-MYC-cry, and pIZ or pIZ-FLAG-jet were immunoprecipitated with antibody to FLAG, and TIM and JET levels were measured. Experiments in each panel were repeated three to five times with similar results. Data are quantified in fig. S3.