Pascal Crottet

Recognition of sorting signals by clathrin adaptors

Sorting of membrane proteins is generally mediated by cytosolic coats, which create a scaffold to form coated buds and vesicles and to selectively concentrate cargo by interacting with cytosolic signals. The classical paradigm is the interaction between clathrin coats and associated adaptor proteins, which cluster receptors with characteristic tyrosine and dileucine motifs during endocytosis. Clathrin in association with different sets of adaptors is found in addition at the trans‐Golgi network and endosomes. Sequences similar to internalization signals also direct lysosomal and basolateral sorting, which implicates related clathrin‐adaptor coats in the respective sorting pathways. This review concentrates on the recognition of sorting signals by clathrin‐associated adaptor proteins, an area of significant recent progress due to new methodological and conceptual approaches.  BioEssays 21:558–567, 1999.

In vivo kinetics of protein targeting to the endoplasmic reticulum determined by site-specific phosphorylation

We have developed a novel assay to detect the cytosolic localization of protein domains by inserting a short consensus sequence for phosphorylation by protein kinase A. In transfected COS‐1 cells, this sequence was labeled efficiently with [32P]phosphate only when exposed to the cytosol and not when translocated into the lumen of the endoplasmic reticulum. The phosphorylation state of this sequence can therefore be used to determine the topology of membrane proteins. This assay is sufficiently sensitive to detect even the transient cytosolic exposure of the N‐terminal domain of a membrane protein with a reverse signal‐anchor sequence. The extent of phosphorylation per newly synthesized polypeptide was shown to reflect the time of exposure to the cytosol, which depends on translation, targeting and translocation of the N‐terminus. By altering the length of the N‐terminal domain or manipulating the translation rate, it was determined that protein targeting is rapid and requires only a few seconds. The rate of N‐terminal translocation was estimated to be ∼1.6 times the rate of translation.

Mapping the Interaction Between Murine IgA and Murine Secretory Component Carrying Epitope Substitutions Reveals a Role of Domains II and III in Covalent Binding to IgA

We have identified sites for epitope insertion in the murine secretory component (SC) by replacing individual surface-exposed loops in domains I, II, and III with the FLAG sequence (Crottet, P., Peitsch, M. C., Servis, C., and Corthésy, B. (1999) J. Biol. Chem. 274, 31445–31455). We had previously shown that epitope-carrying SC reassociated with dimeric IgA (IgAd) can serve as a mucosal delivery vehicle. When analyzing the capacity of SC mutants to associate with IgAd, we found that all domain II and III mutants bound specifically with immobilized IgAd, and their affinity for IgAd was comparable to that of the wild type protein (IC50 ∼ 1 nm). We conclude that domains II and III in SC are permissive to local mutation and represent convenient sites to antigenize the SC molecule. No mutant bound to monomeric IgA. SC mutants exposing the FLAG at their surface maintained this property once bound to IgAd, thereby defining regions not required for high affinity binding to IgAd. Association of IgAd with SC mutants carrying a buried FLAG did not exposede novo the epitope, consistent with limited, local changes in the SC structure upon binding. Only wild type and two mutant SCs bound covalently to IgAd, thus implicating domains II and III in the correct positioning of the reactive cysteine in SC. This establishes that the integrity of murine SC domains II and III is not essential to preserve specific IgAd binding but is necessary for covalency to take place. Finally, SC mutants existing in the monomeric and dimeric forms exhibited the same IgAdbinding capacity as monomeric wild type SC known to bind with a 1:1 stoichiometry.

Interaction of amphiphysins with AP-1 clathrin adaptors at the membrane.

The assembly of clathrin/AP (adaptor protein)-1-coated vesicles on the trans-Golgi network and endosomes is much less studied than that of clathrin/AP-2 vesicles at the plasma membrane for endocytosis. In vitro, the association of AP-1 with protein-free liposomes had been shown to require phosphoinositides, Arf1 (ADP-ribosylation factor 1)-GTP and additional cytosolic factor(s). We have purified an active fraction from brain cytosol and found it to contain amphiphysin 1 and 2 and endophilin A1, three proteins known to be involved in the formation of AP-2/clathrin coats at the plasma membrane. Assays with bacterially expressed and purified proteins showed that AP-1 stabilization on liposomes depends on amphiphysin 2 or the amphiphysin 1/2 heterodimer. Activity is independent of the SH3 (Src homology 3) domain, but requires interaction of the WDLW motif with γ-adaptin. Endogenous amphiphysin in neurons and transfected protein in cell lines co-localize perinuclearly with AP-1 at the trans-Golgi network. This localization depends on interaction of clathrin and the adaptor sequence in the amphiphysins and is sensitive to brefeldin A, which inhibits Arf1-dependent AP-1 recruitment. Interaction between AP-1 and amphiphysin 1/2 in vivo was demonstrated by co-immunoprecipitation after cross-linking. These results suggest an involvement of amphiphysins not only with AP-2 at the plasma membrane, but also in AP-1/clathrin coat formation at the trans-Golgi network.

Recruitment of Coat Proteins to Liposomes and Peptidoliposomes

Intracellular transport within the cell is generally mediated by membrane vesicles. Their formation is typically initiated by activation of small GTPases that then recruit cytosolic proteins to the membrane surface to form a coat, interact with cargo and accessory proteins, and deform the lipid bilayer to produce a transport vesicle. Liposomes proved to be a useful tool to study the molecular mechanisms of these processes in vitro. Here we describe the use of liposomes and peptidoliposomes presenting lipid-coupled cytosolic tails of cargo proteins for the in vitro analysis of the membrane recruitment of AP-1 adaptors in the process of forming AP-1/clathrin coats. AP-1 recruitment is mediated by the GTPase Arf1 and requires specific lipids and cargo signals. Interaction with cargo induces AP-1 oligomerization already in the absence of clathrin. Without cargo peptides, accessory proteins, such as amphiphysin 2, can be identified that stabilize AP-1 binding to liposomal membranes.

Recruitment of Coat Proteins to Peptidoliposomes

Intracellular transport between compartments within the cell is generally mediated by membrane vesicles. Their formation is initiated by activation of small GTPases that then recruit cytosolic proteins to the membrane surface to form a coat, interact with cargo proteins, and deform the lipid bilayer. Liposomes proved to be a useful tool to study the molecular mechanisms of these processes in vitro. To analyze the involvement of membrane proteins, the cytosolically exposed sequences may be coupled chemically to reactive lipids in the membrane. Here we describe the use of such peptidoliposomes presenting lipid-coupled cytosolic tails of cargo proteins for the in vitro analysis of the membrane recruitment of AP-1 adaptors in the process of forming AP-1/clathrin coats. AP-1 recruitment is mediated by the GTPase Arf1, requires specific lipids, and cargo signals. Interaction with cargo induces AP-1 oligomerization already in the absence of clathrin.