Cell Biology - Double agents for mitochondrial division.
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Nature 2016 Nov

McBride HM and Frost A

Mitochondrial organelles — the energy powerhouses of the cell — must divide and fuse dynamically to function. It emerges that two distinct dynamin enzymes enable mitochondrial division.

This preview covers yet another surprising bit of cell biology by Gia Voeltz and was tons of fun to write with Heidi McBride

Membrane fission by dynamin - what we know and what we need to know.
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EMBO J 2016 Nov

Antonny B, Burd C, De Camilli P, Chen E, Daumke O, Faelber K, Ford M, Frolov VA, Frost A, Hinshaw JE, Kirchhausen T, Kozlov MM, Lenz M, Low HH, McMahon H, Merrifield C, Pollard TD, Robinson PJ, Roux A, Schmid S

The large GTPase dynamin is the first protein shown to catalyze membrane fission. Dynamin and its related proteins are essential to many cell functions, from endocytosis to organelle division and fusion, and it plays a critical role in many physiological functions such as synaptic transmission and muscle contraction. Research of the past three decades has focused on understanding how dynamin works. In this review, we present the basis for an emerging consensus on how dynamin functions. Three properties of dynamin are strongly supported by experimental data: first, dynamin oligomerizes into a helical polymer; second, dynamin oligomer constricts in the presence of GTP; and third, dynamin catalyzes membrane fission upon GTP hydrolysis. We present the two current models for fission, essentially diverging in how GTP energy is spent. We further discuss how future research might solve the remaining open questions presently under discussion.

This review paper emerged from the the most delightful scientific event of my life so far: a week of thinking, drinking, debating and discussing at Les Treilles. Special thanks and credit to Aurelien Roux, Oli Duamke, and Pietro De Camilli for leading the effort.

A Golgi rhomboid protease Rbd2 recruits Cdc48 to cleave yeast SREBP.
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EMBO J 2016 Nov

Hwang J, Ribbens D, Raychaudhuri S, Ciarns L, Gu H, Frost A, Urban S, Espenshade PJ

Hypoxic growth of fungi requires sterol regulatory element-binding protein (SREBP) transcription factors, and human opportunistic fungal pathogens require SREBP activation for virulence. Proteolytic release of fission yeast SREBPs from the membrane in response to low oxygen requires the Golgi membrane-anchored Dsc E3 ligase complex. Using genetic interaction arrays, we identified Rbd2 as a rhomboid family protease required for SREBP proteolytic processing. Rbd2 is an active, Golgi-localized protease that cleaves the transmembrane segment of the TatA rhomboid model substrate. Epistasis analysis revealed that the Dsc E3 ligase acts on SREBP prior to cleavage by Rbd2. Using APEX2 proximity biotinylation, we demonstrated that Rbd2 binds the AAA-ATPase Cdc48 through a C-terminal SHP box. Interestingly, SREBP cleavage required Rbd2 binding of Cdc48, consistent with Cdc48 acting to recruit ubiquitinylated substrates. In support of this claim, overexpressing a Cdc48-binding mutant of Rbd2 bypassed the Cdc48 requirement for SREBP cleavage, demonstrating that Cdc48 likely plays a role in SREBP recognition. In the absence of functional Rbd2, SREBP precursor is degraded by the proteasome, indicating that Rbd2 activity controls the balance between SREBP activation and degradation.

This paper has its roots in our systematic genetic studies in fission yeast and relates to our interest in the varied roles of Cdc48. The heavy lifting was all done in the outstanding lab of friend and fellow fission yeast fan Peter Espenshade

Subunit connectivity, assembly determinants and architecture of the yeast exocyst complex.
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Heider MR, Gu M, Duffy CM, Mirza AM, Marcotte LL, Walls AC, Farrall N, Hakhverdyan Z, Field MC, Rout MP, Frost A, Munson M

The exocyst is a hetero-octameric complex that has been proposed to serve as the tethering complex for exocytosis, although it remains poorly understood at the molecular level. Here, we purified endogenous exocyst complexes from Saccharomyces cerevisiae and showed that they are stable and consist of all eight subunits with equal stoichiometry. Using a combination of biochemical and auxin induced-degradation experiments in yeast, we mapped the subunit connectivity, identified two stable four- subunit modules within the octamer and demonstrated that several known exocyst-binding partners are not necessary for exocyst assembly and stability. Furthermore, we visualized the structure of the yeast complex by using negative-stain electron microscopy; our results indicate that the exocyst exists predominantly as a stable, octameric complex with an elongated architecture that suggests that the subunits are contiguous helical bundles packed together into a bundle of long rods.

This paper is our first foray into understanding the fundamental, and fundamentally mysterious, Exocyst complex. Thanks to Mary Munson and Michael Rout for breaking fresh biochemical ground to make structural studies possible.

Structure and membrane remodeling activity of ESCRT-III helical polymers.
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SCIENCE 2015 Dec

McCullough J, Clippinger AK, Talledge N, Skowyra ML, Saunders MG, Naismith TV, Colf LA, Afonine P, Arthur C, Sundquist WI*, Hanson PI *, Frost A*
* Co-corresponding authors

The endosomal sorting complexes required for transport (ESCRT) proteins mediate fundamental membrane remodeling events that require stabilizing negative membrane curvature. These include endosomal intralumenal vesicle formation, HIV budding, nuclear envelope closure, and cytokinetic abscission. ESCRT-III subunits perform key roles in these processes by changing conformation and polymerizing into membrane-remodeling filaments. Here, we report the 4 angstrom resolution cryogenic electron microscopy reconstruction of a one-start, double-stranded helical copolymer composed of two different human ESCRT-III subunits, charged multivesicular body protein 1B (CHMP1B) and increased sodium tolerance 1 (IST1). The inner strand comprises “open” CHMP1B subunits that interlock in an elaborate domain-swapped architecture and is encircled by an outer strand of “closed” IST1 subunits. Unlike other ESCRT-III proteins, CHMP1B and IST1 polymers form external coats on positively curved membranes in vitro and in vivo. Our analysis suggests how common ESCRT-III filament architectures could stabilize different degrees and directions of membrane curvature.

This paper represents a heroic collaboration by scientists from five different universities and is a reminder to me of the power of being open with your colleagues at conferences. Thanks to all, but especially to Wes Sundquist, Phyllis Hanson, Pavel Afonine, and Chris Arthur for making this paper possible. Like our collaborative work on the RQC and the discovery of CAT tails, this paper is another great example of how much fun it is (once the dust from peer review settles) to be surprised by unexpected results!

Thank you to Julie Kiefer for writing up this concise summary with movies for the press.

Structural and functional studies of membrane remodeling machines.
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Kalia R, Talledge N, Frost A

Building a Cell from its Component Parts. Chapter 10. Building cells from their component parts will hinge upon our ability to reconstitute biochemical compartmentalization and exchange between membrane -delimited organelles. By contrast with our understanding of other cellular events, the mechanisms that govern membrane trafficking has lagged because the presence of phospholipid bilayers complicates the use of standard methods. This chapter describes in vitro methods for purifying, reconstituting, and visualizing membrane remodeling activities directly by electron cryomicroscopy.

Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains.
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SCIENCE 2015 Jan

Shen PS, Park J, Qin Y, Li X, Parsawar K, Larson MH, Cox J, Cheng Y, Lambowitz, Weissman JS*, Brandmann O*, Frost A *
* Co-corresponding authors

In Eukarya, stalled translation induces 40S dissociation and recruitment of the ribosome quality control complex (RQC) to the 60S subunit, which mediates nascent chain degradation. Here we report cryo-electron microscopy structures revealing that the RQC components Rqc2p (YPL009C/ Tae2) and Ltn1p (YMR247C/Rkr1) bind to the 60S subunit at sites exposed after 40S dissociation, placing the Ltn1p RING (Really Interesting New Gene) domain near the exit channel and Rqc2p over the P-site transfer RNA ( tRNA). We further demonstrate that Rqc2p recruits alanine- and threonine- charged tRNA to the A site and directs the elongation of nascent chains independently of mRNA or 40S subunits. Our work uncovers an unexpected mechanism of protein synthesis, in which a protein–not an mRNA–determines tRNA recruitment and the tagging of nascent chains with carboxy-terminal Ala and Thr extensions ("CAT tails").

This image was created by Janet Iwasa and illustrates the protein Rqc2 (yellow) coordinating two tRNA moleucles (green and blue) in the PTC of the 60S ribosome without a genetic template. Janet Iwasa’s art was featured in a remarkable number of news outlets highlighting our surprising discovery.

Interchangeable adaptors regulate mitochondrial dynamin assembly for membrane scission.
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Koirala S, Guo Q, Kalia R, Bui HT, Eckert DM, Frost A*, Shaw JM*
* Co-corresponding authors

Mitochondrial fission is mediated by the dynamin-related GTPases Dnm1/Drp1 (yeast/mammals), which form spirals around constricted sites on mitochondria. Additional membrane-associated adaptor proteins (Fis1, Mdv1, Mff, and MiDs) are required to recruit these GTPases from the cytoplasm to the mitochondrial surface. Whether these adaptors participate in both GTPase recruitment and membrane scission is not known. Here we use a yeast strain lacking all fission proteins to identify the minimal combinations of GTPases and adaptors sufficient for mitochondrial fission. Although Fis1 is dispensable for fission, membrane-anchored Mdv1, Mff, or MiDs paired individually with their respective GTPases are sufficient to divide mitochondria. In addition to their role in Drp1 membrane recruitment, MiDs coassemble with Drp1 in vitro. The resulting heteropolymer adopts a dramatically different structure with a narrower diameter than Drp1 homopolymers assembled in isolation. This result demonstrates that an adaptor protein alters the architecture of a mitochondrial dynamin GTPase polymer in a manner that could facilitate membrane constriction and severing activity.

This paper is our first exploration of mitochondria. I’ve enjoyed working on and thinking about the Dynamin-family of larget GTPases since I was a graduate student, but this paper with the delightful Janet Shaw is my lab’s first effort to understand the functional partnership between a large GTPase and one of its binding partners. Stay tuned for round 2!

A ribosome-bound quality control complex triggers degradation of nascent peptides and signals translation stress.
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CELL 2012 Nov

Brandman O, Stewart-Ornstein J, Wong D, Larson A, Williams CC, Li GW, Zhou S, King D, Shen PS, Weibezahn J, Dunn JG, Rouskin S, Inada T, Frost A *, Weissman JS*
* Co-corresponding authors

The conserved transcriptional regulator heat shock factor 1 (Hsf1) is a key sensor of proteotoxic and other stress in the eukaryotic cytosol. We surveyed Hsf1 activity in a genome-wide loss-of-function library in Saccaromyces cerevisiae as well as ~78,000 double mutants and found Hsf1 activity to be modulated by highly diverse stresses. These included disruption of a ribosome-bound complex we named the Ribosome Quality Control Complex (RQC) comprising the Ltn1 E3 ubiquitin ligase, two highly conserved but poorly characterized proteins (Tae2 and Rqc1), and Cdc48 and its cofactors. Electron microscopy and biochemical analyses revealed that the RQC forms a stable complex with 60S ribosomal subunits containing stalled polypeptides and triggers their degradation. A negative feedback loop regulates the RQC, and Hsf1 senses an RQC-mediated translation-stress signal distinctly from other stresses. Our work reveals the range of stresses Hsf1 monitors and elucidates a conserved cotranslational protein quality control mechanism.

After this paper came out and started getting traction, Onn Brandman, Jonathan Weissman and I decided to change the name of Tae2 to Rqc2 for consistency with our subsequent structural and functional work on the RQC and CAT tails.

Functional repurposing revealed by comparing S. pombe and S. cerevisiae genetic interactions.
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CELL 2012 Jun

Frost A*, Elgort MG, Brandman O, Ives C, Collins SR, Miller-Vedam L, Weibezahn J, Hein MY, Poser I, Mann M, Hyman AA, Weissman JS
* corresponding author

We present a genetic interaction map of pairwise measures including ∼40% of nonessential S. pombe genes. By comparing interaction maps for fission and budding yeast, we confirmed widespread conservation of genetic relationships within and between complexes and pathways. However, we identified an important subset of orthologous complexes that have undergone functional "repurposing": the evolution of divergent functions and partnerships. We validated three functional repurposing events in S. pombe and mammalian cells and discovered that (1) two lumenal sensors of misfolded ER proteins, the kinase/nuclease Ire1 and the glucosyltransferase Gpt1, act together to mount an ER stress response; (2) ESCRT factors regulate spindle-pole-body duplication; and (3) a membrane- protein phosphatase and kinase complex, the STRIPAK complex, bridges the cis-Golgi, the centrosome, and the outer nuclear membrane to direct mitotic progression. Each discovery opens new areas of inquiry and-together -have implications for model organism-based research and the evolution of genetic systems.

The cover art for this issue of Cell generated some fun controvsery (and upset lab safety officers everywhere), thanks to the talented Brandon Toyama.

Structural basis of membrane bending by the N-BAR protein Endophilin.
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CELL 2012 Mar

Mim C, Cui H, Gawronski-Salerno JA, Frost A, Lyman E, Voth GA, Unger VM

Functioning as key players in cellular regulation of membrane curvature, BAR domain proteins bend bilayers and recruit interaction partners through poorly understood mechanisms. Using electron cryomicroscopy, we present reconstructions of full-length endophilin and its N-terminal N-BAR domain in their membrane-bound state. Endophilin lattices expose large areas of membrane surface and are held together by promiscuous interactions between endophilin’s amphipathic N-terminal helices. Coarse-grained molecular dynamics simulations reveal that endophilin lattices are highly dynamic and that the N-terminal helices are required for formation of a stable and regular scaffold. Furthermore, endophilin accommodates different curvatures through a quantized addition or removal of endophilin dimers, which in some cases causes dimerization of endophilin’s SH3 domains, suggesting that the spatial presentation of SH3 domains, rather than affinity, governs the recruitment of downstream interaction partners.

Membrane trafficking: decoding vesicle identity with contrasting chemistries.
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Frost A

Proteins involved in membrane traffic must distinguish between different classes of vesicles. New work now shows that α-synuclein and ALPS motifs represent two extreme types of amphipathic helix that are tuned to detect both the curvature of transport vesicles as well as their bulk lipid content.

This essay was inspired by a lovely piece of cell biology by Bruno Antonny and Cathy Jackson.

The F-BAR domain of srGAP2 induces membrane protrusions required for neuronal migration and morphogenesis.
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CELL 2009 Sep

Guerrier S, Coutinho-Budd J, Sassa T, Gresset A, Jordan NV, Chen K, Jin WL, Frost A, Polleux F

During brain development, proper neuronal migration and morphogenesis is critical for the establishment of functional neural circuits. Here we report that srGAP2 negatively regulates neuronal migration and induces neurite outgrowth and branching through the ability of its F-BAR domain to induce filopodia-like membrane protrusions resembling those induced by I-BAR domains in vivo and in vitro. Previous work has suggested that in nonneuronal cells filopodia dynamics decrease the rate of cell migration and the persistence of leading edge protrusions. srGAP2 knockdown reduces leading process branching and increases the rate of neuronal migration in vivo. Overexpression of srGAP2 or its F-BAR domain has the opposite effects, increasing leading process branching and decreasing migration. These results suggest that F-BAR domains are functionally diverse and highlight the functional importance of proteins directly regulating membrane deformation for proper neuronal migration and morphogenesis.

This cover image was created by the first author of this remarkable story, the talented Sabrice Guerrier.

The BAR domain superfamily: membrane-molding macromolecules
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CELL 2009 Apr

Frost A, Unger VM, De Camilli P

Membrane-shaping proteins of the BAR domain superfamily are determinants of organelle biogenesis, membrane trafficking, cell division, and cell migration. An upsurge of research now reveals new principles of BAR domain-mediated membrane remodeling, enhancing our understanding of membrane curvature-mediated information processing.

Structural basis of membrane invagination by F-BAR domains.
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CELL 2008 Mar

Frost A, Perera R, Roux A, Spasov K, Destaing O, Egelman EH, De Camilli P, Unger VM

BAR superfamily domains shape membranes through poorly understood mechanisms. We solved structures of F-BAR modules bound to flat and curved bilayers using electron (cryo)microscopy. We show that membrane tubules form when F-BARs polymerize into helical coats that are held together by lateral and tip-to-tip interactions. On gel-state membranes or after mutation of residues along the lateral interaction surface, F-BARs adsorb onto bilayers via surfaces other than their concave face. We conclude that membrane binding is separable from membrane bending, and that imposition of the module’s concave surface forces fluid-phase bilayers to bend locally. Furthermore, exposure of the domain’s lateral interaction surface through a change in orientation serves as the crucial trigger for assembly of the helical coat and propagation of bilayer bending. The geometric constraints and sequential assembly of the helical lattice explain how F-BAR and classical BAR domains segregate into distinct microdomains, and provide insight into the spatial regulation of membrane invagination.

This painting was created by artist and scientist David Goodsell. The painting was inspired in part by our structures of F-BAR and N-BAR membrane-bound coats, and was commissioned by Min Wu.

F-BAR proteins join the BAR family fold.
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Frost A, De Camilli P, Unger VM

Expanding the range of curvature generating and curvature stabilizing protein modules, the first F-BAR domain structures support their assignment to the BAR domain superfamily and emphasize how modifications to a basic structural frame can generate a broad spectrum of properties.

GTP-dependent twisting of dynamin implicates constriction and tension in membrane fission.
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NATURE 2006 Apr

Roux A, Uyhazi K, Frost A, De Camilli P

Dynamin, a crucial factor in endocytosis, is a member of a family of GTPases that participates in membrane fission. It was initially proposed to act as a machine that constricts and cuts the neck of nascent vesicles in a GTP-hydrolysis-dependent reaction, but subsequent studies suggested alternative models. Here we monitored the effect of nucleotides on dynamin-coated lipid tubules in real time. Addition of GTP, but not of GDP or GTP-gammaS, resulted in twisting of the tubules and supercoiling, suggesting a rotatory movement of the helix turns relative to each other during GTP hydrolysis. Rotation was confirmed by the movement of beads attached to the tubules. Twisting activity produced a longitudinal tension that was released by tubule breakage when both ends of the tubule were anchored. Fission also occurred when dynamin and GTP were added to lipid tubules that had been generated from liposomes by the motor activity of kinesin on microtubules. No fission events were observed in the absence of longitudinal tension. These findings demonstrate a mechanoenzyme activity of dynamin in endocytosis, but also imply that constriction is not sufficient for fission. At the short necks of endocytic vesicles, other factors leading to tension may cooperate with the constricting activity of dynamin to induce fission.

Click for a full listing of Adam Frost's publications