C ONFLICTOS SOCIALES DESARROLLADOS EN MÁS DE UN DEPARTAMENTO

187

Molecular organization and function of the Golgi stacks.

Y. Xiang1, X. Zhang1, Y. Wang1; 1University of Michigan, Ann Arbor, MI

The unique structure of the Golgi in almost all eukaryotic cells is a stack of flattened cisternal membranes, but how this structure is formed at the molecular level and why its formation is important for cellular functions remain elusive. We have developed an in vitro system to reconstitute the process of mitotic Golgi disassembly and post-mitotic reassembly in mammalian cells, which allowed us to reveal the molecular mechanism of the Golgi biogenesis during cell division. Mitotic Golgi fragmentation involves membrane vesiculation coupled with cisternal unstacking; post-mitotic Golgi reassembly is mediated by membrane fusion to form single cisternae and stack formation. Stack formation directly involves the Golgi stacking protein

GRASP65 and GRASP55, which play complementary and essential roles in Golgi cisternal stacking by forming mitotically regulated trans-oligomers. By depletion of GRASP65/55 we are able to manipulate Golgi stack formation and thus determine the biological significance of stacking. We demonstrate that Golgi cisternal unstacking stimulates COPI vesicle budding and thus enhances protein transport. Golgi fragmentation, however, impairs protein sorting and alters the glycosylation of cell surface proteins and reduces cell adhesion. Subsequently, cell adhesion and migration were reduced when the Golgi was unstacked, which was probably the result of reduced ¦Á5/¦Â1 integrin expression. Furthermore, total protein synthesis and the proliferation of cells with unstacked Golgi were enhanced. Inhibition of Golgi disassembly at the onset of mitosis also affects cell cycle progression. We propose that Golgi stack formation is a flux regulator for protein trafficking and thereby functions as a quality control mechanism for protein sorting and modification. Structural and functional Golgi defects in disease models are explored in this study.

188

ER tubules mark sites of mitochondrial division.

J. Friedman1, L. Lackner2, M. West1, J. DiBenedetto1, J. Nunnari2, G. Voeltz1; 1University of Colorado, Boulder, Boulder, CO, 2University of California, Davis, CA

Mitochondrial structure and distribution are regulated by division and fusion events. Mitochondrial division is regulated by Dnm1/Drp1, a dynamin-related protein that forms helices around mitochondria to mediate fission. Little is known about what determines sites of mitochondrial fission within the mitochondrial network. Given that ER and mitochondria exhibit tightly coupled dynamics and have extensive contacts, we tested whether ER plays a role in mitochondrial division. We show that mitochondrial division occurs at positions where ER tubules contact mitochondria and mediate constriction prior to Drp1 recruitment. These data demonstrate that ER tubules play an active role in defining the position of mitochondrial division sites.

189

Coordinating Mitochondrial Biogenesis and Redistribution to Achieve Proper Inheritance of Mitochondrial Content in Budding Yeast.

S. Rafelski1, M. Viana2, L. Costa2, W. Marshall1; 1Biochemistry, UC San Francisco, San Francisco, CA, 2Inst. de Fisica de Sao Carlos, USP, Sao Carlos, Brazil

Mitochondria must grow with the growing cell to ensure proper cellular physiology and inheritance upon division. But it is unknown how mitochondrial content scales with cell size during asymmetric cell growth and division. Is the same mitochondrial to cell size ratio maintained uniformly throughout the growing cell to produce identical mother and daughter cells? Or is there an asymmetry in the inheritance of mitochondrial content? We previously developed a novel computational method that quantifies mitochondrial networks as 3D mathematical graphs to measure the physical size of the mitochondrial network. Using this method, we found mitochondrial inheritance to be very asymmetric. Regardless of mother size or mitochondrial content, all buds attained the same average mitochondrial to cell size ratio with the same kinetics during budding. In contrast, aging mothers experienced a continued decrease in their mitochondrial to cell size ratio over successive generations. The proper mitochondrial volume ratio in the bud must is generated by a combination of net biogenesis (sum of new synthesis and turnover) in each, and net redistribution (sum of all movement and retention of tubules) between, mother and bud compartments. We calculated the relative contributions of net biogenesis and redistribution to the mitochondrial content in the bud. We found that redistribution dominated during the first half of budding and biogenesis in the bud took over

during the second half. A final 60-75% of the mitochondria inherited by the buds originated in their mothers. Mitochondrial biogenesis in the mothers could not keep up with the required redistribution into their buds. In the youngest, first generation mothers decreased redistribution was compensated for by increased biogenesis in their buds to achieve the same volume ratio as the rest of the population. We delayed or sped up mitochondrial inheritance with deletion or overexpression of Ypt11p and Mmr1p, representing two of the three mitochondrial inheritance pathways. In both cases the kinetics of mitochondrial inheritance were significantly altered but buds still reached the proper mitochondrial volume ratio in time for division. We conclude that cells actively regulate the mitochondrial volume ratio in their buds by regulating both redistribution and biogenesis to ensure proper inheritance of mitochondrial content.

190

mTORC1 Senses Amino Acids Through a Lysosomal Inside-Out Mechanism that Requires the Vacuolar H+-ATPase.

R. Zoncu1, L. Bar Peled1, A. Efeyan1, S. Wang1, Y. Sancak1, D. Sabatini1; 1Whitehead Institute for Biomedical Research, Cambridge, MA

The mTOR Complex 1 (mTORC1) kinase is a master growth regulator that senses amino acids through a poorly understood mechanism. A key event is the amino acid-induced activation of the Rag GTPases, which promotes the translocation of mTORC1 to the lysosomal surface, the site of mTORC1 activation. A central question is where in the cell amino acids are sensed and how their sensing leads to Rag activation. Here, we identify the vacuolar H+-ATPase (v- ATPase) as necessary for amino acid signaling to mTORC1. The v-ATPase functions upstream of the Rag GTPases and promotes amino acid-mediated recruitment of mTORC1 to the lysosomal surface. Using an assay that recapitulates amino acid signaling to mTORC1 in a cell- free system, we show that the catalytic movement of the v-ATPase, but not the lysosomal pH gradient, is necessary for amino acids to regulate the Rag GTPases and promote mTORC1 translocation. Moreover, using this system we implicate the lysosomal lumen as the site where amino acid signaling initiates. These results identify the v-ATPase as a new component of the mTOR pathway and delineate a lysosome-associated machinery for amino acid sensing.

191

The interaction of MiT/TFE transcription factors with lysosomes contributes to regulation of lysosomal homeostasis.

A. Roczniak-Ferguson1, C. Petit1, S. Qian1, S. Ferguson1; 1Department of Cell Biology, Yale University, New Haven, CT

The MiT/TFE family of transcription factors (MITF, TFEB, TFE3) regulates expression of many genes encoding proteins involved in autophagy and the biogenesis of lysosomes and lysosome- related organelles. In this study, we show that the regulation of MiT/TFE transcription factor abundance in the nucleus is tightly linked to autophagy-inducing stimuli and overall lysosomal status and that this regulation depends on a physical interaction of these transcription factors with lysosomes via a conserved region at their amino terminus. This lysosomal recruitment promotes MiT/TFE phosphorylation within a motif that confers a phosphorylation-dependent interaction with 14-3-3 proteins. This interaction retains MiT/TFE proteins mostly in the cytoplasm under basal conditions. In response to specific autophagy-inducing stimuli and/or perturbation of lysosomal function, phosphorylation of the14-3-3 binding site is attenuated, the interaction with 14-3-3 proteins is lost and the MiT/TFE proteins accumulate in the nucleus. Building on the previously established ability of these transcription factors to up-regulate the expression of genes within the autophagy-lysosomal pathway, our data suggests a novel homeostatic feedback mechanism that couples demand for lysosomal activity to the

transcriptional control of the expression of genes encoding proteins critical for the function of this organelle.

192

The PX-BAR protein SNX18 is required for autophagy.

H. Knævelsrud1, K. Søreng1, K. Håberg2, F. Rasmuson2, C. Raiborg3, K. Liestøl3, H. Stenmark3, S. Carlsson2, A. Simonsen1; 1Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway, 2Department of Medical Biochemistry and Biophysics, Umeå university, Sweden,

3_{Centre for Cancer Biomedicine, University of Oslo, Oslo, Norway}

Autophagy is a catabolic pathway targeting cytoplasmic material for lysosomal degradation, thereby protecting cells from accumulation of toxic components and enabling cells to survive scarce nutrient supplies. Macroautophagy is characterized by the formation of double-

membraned vesicles, but the membrane remodeling events required for formation of autophagic vesicles are still not completely understood. However, the class III PI3K/Vps34 complex and phosphoinositol-3-phosphate are of core importance to induction of autophagy. Since PX domain proteins are known to bind PI3P and other phosphoinositides and mediate membrane remodeling and trafficking events, we performed an imaging-based siRNA screen targeting all human PX domain proteins using GFP-LC3 autophagosome formation as a read-out and found depletion of the PX-BAR protein SNX18 to strongly inhibit autophagosome formation.

Consequently, overexpression of SNX18 increases LC3 lipidation and GFP-LC3 spot formation and we demonstrate that membrane binding of SNX18 is required for efficient autophagosome formation. Moreover, SNX18 colocalizes with and interacts with the autophagy-associated proteins LC3 and TBK1. In conclusion, our study identified the PX-BAR protein SNX18 to be involved in membrane events required for autophagosome formation.