CNR - Institute of Neuroscience CNR
Institute of Neuroscience
 

Project

Membrane biogenesis and organelle architecture

One of the most intriguing problems in modern Cell Biology is how eukaryotic cells build their organelles, endowing each one with its specific cohort of proteins and lipids. My group is interested in unravelling the mechanisms by which newly synthesized membrane proteins insert into lipid bilayers, how membrane proteins are sorted to different compartments of the secretory pathway, and what determines the extension and architecture of organelles.

Mechanisms of insertion of membrane proteins into the lipid bilayer

Most membrane proteins are co-translationally inserted into the Endoplasmic Reticulum (ER) via the extensively investigated Sec61 protein-conducting channel. An exception to this rule is the insertion of so-called tail-anchored (TA) proteins. These proteins have an N-terminal functional domain in the cytosol and a membrane-anchoring domain very close to the C-terminus, which emerges from the ribosome only upon chain termination. TA proteins are involved in fundamental aspects of cell physiology, such as regulation of apoptosis (bcl-2 proteins), membrane fusion (SNARE proteins), protein translocation (e.g., subunits of the Sec61 complex). Because of the location of their transmembrane domain, their insertion into the phospolipid bilayer is necessarily post-translational.

To investigate the mechanism of insertion of TA proteins into the ER membrane, we have developed stringent assays, based on protection from proteolysis of the C-terminal residues after translocation across the bilayer and on glycosylation of a consensus site engineered at the C-terminus of TA substrates. We have combined diverse experimental approaches, including expression of substrates in yeast cells defective in the known translocation pathway and translocation assays on the in vitro synthesized protein.

Our results establish conclusively that TA protein insertion does not require the Sec61 pathway, either in vivo or in vitro. In attempting to identify the molecules involved in the insertion, we have now defined two classes of TA proteins: those with only moderately hydrophobic transmembrane domain (TMD) (type I TA proteins) are capable of translocation across protein-free bilayers without assistance from any membrane or cytosolic protein; those with more strongly hydrophobic TMDs, (type II) require a novel chaperone system (the GET system) which is presently being investigated by a number of laboratories. We believe that these requirements are linked to problems of delivery of the class II TA substrate to the bilayer in a translocation-competent form, rather than in the translocation step itself. Very interestingly, we find that members of the first class of TA proteins are able to translocate surprisingly long polar domains across the bilayer (up to 100 residues), a finding that we believe is relevant to membrane evolution, biogenesis, and physiology.

Our future goal is to define effect of folding of the polar domain translocated without assistance by type I TA proteins. We are also attempting to define the molecular requirements for insertion of class II substrates in mammalian cells and the mechanism by which some TA proteins avoid the ER and are targeted instead to the outer mitochondrial membrane.

Mechanisms of sorting at the ER/Golgi interface

Complex sorting events occur at ER exit sites, so that the ER maintains its cohort of membrane and lumenal proteins while allowing transport of cargo proteins down the secretory pathway. Much effort has been dedicated to deciphering export signals on cargo proteins and retrieval signals that permit resident proteins to escape from outgoing traffic. In our group, we have focussed on a different feature of transmembrane proteins that affects their sorting behaviour. We have found that the transmembrane domain (TMD) length and hydrophobicity is the main feature determining the ER residence of our model TA protein, cytochrome b5. b5 mutants, with lengthened and more hydrophobic TMDs escape from the ER and reach the plasma membrane.

During the past years, we have been investigating the mechanism of the TMD-dependent sorting. To study sorting between short- and long TMD TA proteins at the very early steps of the secretory pathway, we microinject cells with GFP-tagged constructs and observe transport in live cells. Our results demonstrate that sorting between 2 TA constructs with different TMD length occurs already at the ER.

 

At short times after expression, the two proteins can be seen to occupy different domains of the ER; subsequently, the long TMD protein accumulates at exit sites, while the construct with the short TMD is excluded from these sites. Thus, TMD-dependent sorting occurs at the earliest steps of the secretory pathway, with features consistent with a partitioning mechanism.

The TMD-dependant sorting that we have characterized does not depend on any particular sequence, but rather is dependent on the physical chemical properties of the TMD. We hypothesize that it may depend on interactions with differently sorted lipids, or by different partitioning of TMDs into differently curved ER domains (tubules, cisternae or exit sites). Hence, a self-organizing principle may underlie TMD-dependent sorting. Indeed, by differential scanning calorimetry, we have demonstrated in a model system that two TA protein variants, differing in TMD length, interact differently with lipids. Our future goal is to directly probe with a cross-linking approach the interaction of these differently sorted TA proteins with lipids, and to investigate their curvature-dependent partitioning in model systems.

Factors determining Endoplasmic Reticulum architecture

The ER is a strikingly plastic organelle, being able to adapt its size, organization, and molecular composition to a changing environment. A complex signalling pathway, known as Unfolded Proteins Response (UPR), by which the ER responds to an altered load in its lumen has been extensively characterized. Less is known on the mechanisms by which it responds to an altered load of membrane proteins, and on the factors that determine the segregation of rough and smooth domains of this organelle.

We have investigated changes in ER organization by a combination of electron microscopy and live cell imaging techniques after expressing fluorescently labelled TA proteins targeted to the ER. By establishing a cell line inducibly expressing an ER-targeted TA protein, we have generated a tool with which to investigate the molecular pathways involved in ER expansion induced by ER membrane proteins. Indeed, induction of the expression of the TA reporter at very modest levels (≈ 1% of ER proteins), results in a four fold stimulation of phospholipid synthesis. This system has allowed us to analyse the signalling pathways involved in the response. Very interestingly, we find that only one of the three sensors of the UPR, the transcriptional factor ATF6, is slectively activated. These results suggest that distinct mechanisms can operate to modulate the UPR with consequent different outputs of this signalling pathway. We are presently investigating the mechanism by which increased ER membrane protein expression activates ATF6.

We have recently become interested in changes in ER structure occurring under pathological conditions. In this context, we have been investigating the effects of a mutated ER protein linked to a familial form of amyotrophic lateral sclerosis (P56S-VAPB). Our results show that the mutant protein causes dramatic restructuring of the ER, yet the cell can clear the mutant VAPB inclusions remarkably rapidly by an ERAD-related process. With cell culture models, we are not attempting to unravel the pathogenic mechanism of this dominantly inherited, disease-linked gene.

Publications

  • Maiuolo J, Bulotta S, Verderio C, Benfante R, Borgese N (2011) Selective activation of the transcription factor ATF6 mediates endoplasmic reticulum proliferation triggered by a membrane protein. Proc. Natl. Acad. Sci. U.S.A. 108:7832-7.
  • Borgese N, Fasana E (2011) Targeting pathways of C-tail-anchored proteins. Biochim. Biophys. Acta 1808:937-46.
  • Borgese N, Righi M (2010) Remote origins of tail-anchored proteins. Traffic 11:877-85.
  • Fasana E, Fossati M, Ruggiano A, Brambillasca S, Hoogenraad CC, Navone F, Francolini M, Borgese N (2010) A VAPB mutant linked to amyotrophic lateral sclerosis generates a novel form of organized smooth endoplasmic reticulum. FASEB J. 24:1419-30.
  • Colombo SF, Longhi R, Borgese N (2009) The role of cytosolic proteins in the insertion of tail-anchored proteins into phospholipid bilayers. J. Cell. Sci. 122:2383-92.
  • Ronchi P, Colombo S, Francolini M, Borgese N (2008) Transmembrane domain-dependent partitioning of membrane proteins within the endoplasmic reticulum. J. Cell Biol. 181:105-18.
  • Borgese N, Brambillasca S, Colombo S (2007) How tails guide tail-anchored proteins to their destinations. Curr. Opin. Cell Biol. 19:368-75.
  • Brambillasca S, Yabal M, Makarow M, Borgese N (2006) Unassisted translocation of large polypeptide domains across phospholipid bilayers. J. Cell Biol. 175:767-77.
  • Borgese N, Francolini M, Snapp E (2006) Endoplasmic reticulum architecture: structures in flux. Curr. Opin. Cell Biol. 18:358-64.

Grants

AIRC (Associazione Italiana per la Ricerca sul Cancro / Italian Association for Cancer Research), Milan, Italy.

Fondazione Cariplo / Cariplo Foundation, Milan, Italy.

Collaborations

  • Britta Bruegger, Felix Wieland, University of Heidelberg, Germany.
  • Bruno Goud, Institut Curie, Paris, France.
  • M. Masserini, Dip. di Medicina Sperimentale, Università di Milano Bicocca, Milan, Italy.
  • Renato Longhi, CNR Institute of Molecular Recognition, Milan, Italy.
  • Francesca Navone, CNR Institute of Neuroscience, Milan, Italy.
  • Maura Francolini, CNR Institute of Neuroscience, Milan, Italy.

 

PI photo

Nica Borgese

Contact information

email  E-mail

email  +39 02 5031 6971

Participating staff

Sara Colombo

Elisa Fasana

Jessica Maiuolo

Patrizia Cassella

Matteo Fossati

Stefania Bulotta