TMEM74, a lysosome and autophagosome protein, regulates autophagy.

Autophagy is an intracellular degradation/recycling process in eukaryotic cells. It contributes to the turnover of cellular components by delivering portions of the cytoplasm and organelles to lysosomes for digestion. The molecular mechanisms of autophagy and vesicle trafficking, especially the biogenesis and turnover of autophagosomes, are poorly understood. In this report, we describe the biological activity of a novel autophagy-related molecule, FLJ30668, or Transmembrane protein 74 (TMEM74). Its transcript was identified by Northern blot and the open reading frame was found to encode 393 amino acids, which shared very little identity with other genetic products. Subcellular localization analysis showed TMEM74 localized to the lysosome and autophagosome. Overexpression of TMEM74 in HeLa cells resulted in autophagic vacuolization, increased the dotted distribution of MDC and GFP-LC3, and endogenous LC3-II levels. Wortmannin, an autophagy inhibitor, partially attenuated these effects. Moreover, knockdown of TMEM74 by small interference RNA abolished the autophagic characteristics induced by starvation. These findings demonstrate that TMEM74 may be involved in promoting functional autophagy during cell starvation and other stress conditions.

[Cloning, expression and subcellular localization of a novel human gene-RNF122].

OBJECTIVE: To clone a functionally unknown gene RNF122 and analyze its expression pattern and subcellular localization. METHODS: PCR was used to clone the novel gene-RNF122 from the mixed human tissue cDNA library. Bioinformatics analysis was used to identify structure characteristics of the gene. Northern blot was used to analyze its expression in normal tissues; RT-PCR was employed for its expression in cell lines and tumor tissues. By co-focal microscope, we identified its subcellular localization with organelle markers. RESULTS: A novel human gene-RNF122 was cloned, which was proved to have been expressed in several normal and tumor tissues and many cell lines, and localized in ER and Golgi apparatus. CONCLUSION: RNF122 is a novel gene which encodes a protein that has a classic RING domain. It is widely expressed in several tissues and cell lines. The encoded protein is localized in ER and Golgi apparatus, which indicates that it may play a role in the process of protein degradation.

[Effect of anti-midgut-protein-ingredient antibodies of Anopheles stephensi on the oocysts of Plasmodium yoelii].

OBJECTIVE: To observe the inhibitory effect of the antibodies against midgut-protein-ingredient of Anopheles stephensi on the oocysts of Plasmodium yoelii. METHODS: Female An. stephensi mosquitoes raised in laboratory were dissected and the midguts were collected. Eight BALB/c mice were immunized using midgut-protein (100 microg/mouse, 4 times with an interval of 7-10 day). Ten days after the last immunization, blood was taken from mice armpit artery and serum separated. The immune active antigen of the midgut protein was analyzed by Western blotting. Protein with Mr 38 000-50 000 was separated by sephadex filtering and used to immunize 12 BALB/c mice (100 microg/ mouse, 4 times with interval of 7-10 days). PBS control group was established. Seven days after the last immunization, serum antibody was detected by ELISA. When the antibody titer in immunized mice reached > or = 1:2 560, mice in both groups were infected by P. yoelii (about 2 x 10(7) plasmodium-infected RBC) by abdominal injection. The mosquitoes were fed on the infected mice when the number of female gametes was higher than 2 per 10 microscopical fields 3 days later. After 9 days, the mosquitoes were dissected and the amount of oocysts in midgut was counted. RESULTS: Eight protein bands were shown in midgut-protein of An. stephensi by Western blotting and the band of Mr 38 000-50 000-midgut-protein appeared clearer. The infection rate of oocysts in the experiment and control groups were 28.70% (62/216) and 51.09% (47/92) respectively (P<0.05), with an oocyst index of 14.14 (1 541/109) and 26.02 (1 223/47) respectively (P<0.01). CONCLUSIONS: The midgut protein of Anopheles stephensi with Mr 38 000-50 000 has immune activity, and the antibodies against this protein shows an inhibitory effect on the development of oocysts of Plasmodium yoelii.