CNR - Institute of Neuroscience CNR
Institute of Neuroscience


Insertion and trafficking of membrane proteins into intracellular membranes: mechanisms of insertion into the ER membrane

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.

Assembly and trafficking of nicotinic receptors

Ionotropic neuronal nicotinic acetylcholine receptors (nAChRs) are a heterogeneous class of cationic channels widely distributed in the nervous system whose opening is controlled by the endogenous neurotransmitter acetylcholine (ACh). They consist of five subunits and their different subunit composition generates distinct receptor subtypes with particular functional responses to ACh and drugs. The properties of nAChRs and their role in cell functions depend on their subunit content and their localization. Like other multimeric ion channels, nicotinic receptors undergo particularly stringent quality control to ensure correct folding and subunit assembly at the level of the endoplasmic reticulum (ER). Once the receptor is properly folded, it can leave the ER and through the secretory pathway it can reach the plasma membrane.

In order to study the assembly of the pentameric form of the receptor, the exit from the endoplasmic reticulum membrane and its arrival at the plasma membrane we transfected Hela cells with nicotinic receptor subunits. In this reconstituted system we studied the trafficking of a specific subtype of nicotinic receptor, made of alpha3 and beta4 subunits, its degradation by the proteasome and its regulation by nicotine.

Recent human genetic studies revealed a link between these specific subunits, whose genes are clustered with that of the accessory subunit alpha5, nicotine dependence phenotypes and lung cancer. In particular it was found that several single-nucleotide polymorphisms (SNPs) are associated with smoking-related behaviors. One of these SNPs leads to an asparagine-to-aspartic acid substitution in the nicotinic receptor alpha5 subunit at the amino acid position 398

In our reconstituted system we will investigate the role of the alpha5 subunit in the trafficking of the alpha3beta4 subtype and whether the presence of the polymorphism at the amino acid in position 398 can affect the level and /or the trafficking of the alpha3beta 4 subtype.


  • 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.


PI photo

Sara Colombo

Contact information

email  E-mail

email  +39 02 50316971

Participating staff

Nica Borgese

Patrizia Cassella

Cecilia Gotti

Francesca Mazzo