Arabidopsis thaliana hit1-1 is a heat-intolerant mutant. The HIT1 gene encodes a protein that is homologous to yeast Vps53p, which is a subunit of the Golgi-associated retrograde protein (GARP) complex that is involved in retrograde membrane trafficking to the Golgi. To investigate the correlation between the cellular role of HIT1 and its protective function in heat tolerance in plants, it was verified that HIT1 was co-localized with AtVPS52 and AtVPS54, the other putative subunits of GARP, in the Golgi and post-Golgi compartments in Arabidopsis protoplasts. A bimolecular fluorescence complementation assay showed that HIT1 interacted with AtVPS52 and AtVPS54, which indicated their assembly into a protein complex in vivo. Under heat stress conditions, the plasma membrane of hit1-1 was less stable than that of the wild type, as determined by an electrolyte leakage assay, and enhanced leakage occurred before peroxidation injury to the membrane. In addition, the ability of hit1-1 to survive heat stress was not influenced by exposure to light, which suggested that the heat intolerance of hit-1 was a direct outcome of reduced membrane thermostability rather than heat-induced oxidative stress. Furthermore, hit1-1 was sensitive to the duration (sustained high temperature stress at 37 °C for 3 d) but not the intensity (heat shock at 44 °C for 30 min) of exposure to heat. Collectively, these results imply that HIT1 functions in the membrane trafficking that is involved in the thermal adaptation of the plasma membrane for tolerance to long-term heat stress in plants.
The development of juvenile seedlings after germination is critical for the initial establishment of reproductive plants. Ethylene plays a pivotal role in the growth of seedlings under light or dark during early development. Previously, we identified small molecules sharing a quinazolinone backbone that suppressed the constitutive triple response phenotype in dark-grown eto1-4 seedlings. We designated these small molecules as ACSinhibitor quinazolinones (acsinones), which were uncompetitive inhibitors of 1-aminocyclopropane-1-carboxylic acid synthase. To explore the additional roles of acsinones in plants, we screened and identified 19 Arabidopsis mutants with reduced sensitivity to acsinone7303, which were collectively named revert to eto1 (ret) because of their recovery of the eto1 phenotype. A map-based cloning approach revealed that CELLULOSE SYNTHASE6 (CESA6) and DE-ETIOLATED2 (DET2) were mutated in ret8 (cesa6(ret8);eto1-4) and ret41 (det2(ret41);eto1-5), respectively. Etiolated seedlings of both ret8 and ret41 exhibit short hypocotyls and roots. Ethylene levels were similar in etiolated cesa6(ret8) and det2-1 and in eto1 mutants treated with acsinone7303. Chemical inhibitors of ethylene biosynthesis and perception did not significantly suppress the etiolated phenotype of cesa6(ret8) and det2(ret41). However, together with eto1, cesa6(ret8) and det2(ret41) exhibited an enhanced phenotype in the hypocotyls and apical hooks of etiolated seedlings. These results confirm that, in addition to ethylene, cellulose synthesis and brassinolides can independently contribute to modulate hypocotyl development in young seedlings.
Glioblastoma multiforme (GBM) is the most common and lethal type of brain cancer. Proteasome inhibitors are emerging as a new class of anti-glioma agents; however, the mechanisms of their killing malignant cells are still unclear. We treated U87MG cells with the proteasome inhibitor MG132 and found that cell death correlated with caspase-8 activation and autophagy protein p62/SQSTM1.To explore the role of autophagy and p62/SQSTM1 in MG132-induced cancer cell death, we measured the alteration of MG132's cytotoxicity by autophagy inhibition, autophagy induction or variation of p62/SQSTM1 gene expression. Autophagy was activated upon MG132 treatment for short periods, while inhibition of autophagy aggravated MG132-induced cell death followed by high levels of p62/SQSTM1 and active caspase-8 (p18). Moreover, U87MG cell death was dependent on p62/SQSTM1, and its function required its C-terminus UBA domain to attenuate the MG132-induced cell death. The results suggest that p62/SQSTM1 is a potential contributor in determining the fate of U87MG cells deficient in proteolytic activity.
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