Alpha Tonoplast Intrinsic Protein is Specifically Associated with Vacuole Membrane Involved in an Autophagic Process

Moriyasu, Yuji; Hattori, Masaki; Jauh, Guang‐Yuh; Rogers, John C.

doi:10.1093/pcp/pcg100

Cited by 69 publications

(48 citation statements)

References 19 publications

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“…Finally, plant root tips cultured in nutrient-sufficient medium display constitutive autophagic flux (i.e., a basal level), which is enhanced in nutrient-deprived medium. 696,778,779 Conclusion: The levels of basal autophagy can vary substantially and can mask the effects of the experimental parameters being tested. Changes in media and growth conditions need to be examined empirically to determine affects on basal autophagy and the appropriate times for subsequent manipulations.…”

Section: 696mentioning

confidence: 99%

Guidelines for the use and interpretation of assays for monitoring autophagy

Klionsky¹,

Abdalla²,

Abeliovich³

et al. 2012

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In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

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Section: 696mentioning

confidence: 99%

Guidelines for the use and interpretation of assays for monitoring autophagy

Klionsky¹,

Abdalla²,

Abeliovich³

et al. 2012

Self Cite

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“…As our current data demonstrated that PHO1 is not mainly destined for PM localization and that PHO2-dependent PHO1 degradation requires the MVB-mediated vacuolar degradation pathway as revealed by the E-64d treatment ( Figures 12A and 12B), we inferred that the population of PHO1 subject to vacuolar proteolysis is derived from the EM rather than the PM pools. Interestingly, it was shown that treatment of barley (Hordeum vulgare) and Arabidopsis root tips with E-64d led to the accumulation of undegraded cytoplasmic components in the lytic compartments for autophagy (Moriyasu et al, 2003;Inoue et al, 2006). Since autophagy plays a pivotal role in responses to nutrient deprivation, it is counterintuitive to consider its involvement in degradation of PHO1 when Pi is adequate.…”

Section: Pho2-dependent Turnover Of Pho1 In the Emsmentioning

confidence: 99%

PHO2-Dependent Degradation of PHO1 Modulates Phosphate Homeostasis in Arabidopsis

et al. 2012

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The Arabidopsis thaliana pho2 mutant, which is defective in a ubiquitin-conjugating E2 enzyme, displays inorganic phosphate (Pi) toxicity as a result of enhanced uptake and root-to-shoot translocation of Pi. To elucidate downstream components of the PHO2-dependent regulatory pathway, we identified two pho2 suppressors as carrying missense mutations in PHO1, which has been implicated in Pi loading to the xylem. In support of the genetic interaction between PHO1 and PHO2, we found that the protein level of PHO1 is increased in pho2, whereas such accumulation is ameliorated in both pho2 suppressors. Results from cycloheximide and endosomal Cys protease inhibitor E-64d treatments further suggest that PHO1 degradation is PHO2 dependent and involves multivesicular body-mediated vacuolar proteolysis. Using the transient expression system of tobacco (Nicotiana tabacum) leaves, we demonstrated that PHO1 and PHO2 are partially colocalized and physically interact in the endomembranes, where the ubiquitin conjugase activity of PHO2 is required for PHO1 degradation. In addition, reduced PHO1 expression caused by PHO1 mutations impede Pi uptake, indicating a functional association between xylem loading and acquisition of Pi. Together, our findings uncover a pivotal molecular mechanism by which PHO2 modulates the degradation of PHO1 in the endomembranes to maintain Pi homeostasis in plants.

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“…To study the correlation between vacuole development and stomatal movement, first we used a membrane-permeable Cys protease inhibitor, (2s,3s)-trans-epoxy-succinyl-L-leucylamido-3-methylbutane ethyl ester (E-64d). Application of E-64d to the root cells of barley (Hordeum vulgare) caused the accumulation of starvationinduced autolysosomes (Moriyasu et al, 2003) or N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl) hexatrienyl) pyridinium dibromide-stainable small vesicles surrounding the nucleus (Yamada et al, 2003) in cultured tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) suspension cells in a Suc-free condition. These results showed that E-64d could inhibit the fusion of endosomes with vacuoles (Yamada et al, 2003(Yamada et al, , 2005.…”

Section: Fusion Of Small Vacuoles Was Correlated To Stomatal Openingmentioning

confidence: 99%

The Dynamic Changes of Tonoplasts in Guard Cells Are Important for Stomatal Movement inVicia faba

et al. 2005

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Stomatal movement is important for plants to exchange gas with environment. The regulation of stomatal movement allows optimizing photosynthesis and transpiration. Changes in vacuolar volume in guard cells are known to participate in this regulation. However, little has been known about the mechanism underlying the regulation of rapid changes in guard cell vacuolar volume. Here, we report that dynamic changes in the complex vacuolar membrane system play a role in the rapid changes of vacuolar volume in Vicia faba guard cells. The guard cells contained a great number of small vacuoles and various vacuolar membrane structures when stomata closed. The small vacuoles and complex membrane systems fused with each other or with the bigger vacuoles to generate large vacuoles during stomatal opening. Conversely, the large vacuoles split into smaller vacuoles and generated many complex membrane structures in the closing stomata. Vacuole fusion inhibitor, (2s,3s)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester, inhibited stomatal opening significantly. Furthermore, an Arabidopsis (Arabidopsis thaliana) mutation of the SGR3 gene, which has a defect in vacuolar fusion, also led to retardation of stomatal opening. All these results suggest that the dynamic changes of the tonoplast are essential for enhancing stomatal movement.

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Alpha Tonoplast Intrinsic Protein is Specifically Associated with Vacuole Membrane Involved in an Autophagic Process

Cited by 69 publications

References 19 publications

Guidelines for the use and interpretation of assays for monitoring autophagy

Guidelines for the use and interpretation of assays for monitoring autophagy

PHO2-Dependent Degradation of PHO1 Modulates Phosphate Homeostasis in Arabidopsis

The Dynamic Changes of Tonoplasts in Guard Cells Are Important for Stomatal Movement inVicia faba

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