LITAF: coupling ubiquitination to transport at the endosome

How cells respond to their environment is determined by hundreds of different membrane proteins that are expressed on the cell surface. Cells must constantly sample and modify this complement of surface membrane proteins, to remove proteins that are no longer required at the surface due to a changed environment, or to replace proteins which become damaged by day-to-day stresses. The process that performs this important 'quality control' is endocytosis, whereby surface membrane proteins are internalised ('endocytosed') within vesicles to reach an intracellular compartment, the endosome. From there, membrane proteins that are no longer required are packaged into internal vesicles within the endosome, and the resulting structure (the multivesicular body) is then transported towards a degradative compartment (the lysosome). Alternatively, surface membrane proteins that are to be re-used are packaged at the endosome into membrane tubules and return to the cell surface. Each step of the endosomal pathway involves machinery that selects cargo membrane proteins for different destinations, coupled to machinery that deforms the membrane to generate transport intermediates (i.e. vesicles and tubules). In summary, the endosomal pathway is characterised by an abundance of high-curvature membranes that enact transport reactions.

The function of virtually all cellular proteins is regulated by post-translational modifications. One key such modification is ubiquitination (the covalent attachment of the small protein ubiquitin, or polyubiquitin chains). Ubiquitination is widespread, with cells expressing >1000 ubiquitinating enzymes (ubiquitin ligases), but is particularly important during endocytosis, where it performs several important tasks. First, membrane proteins that are targeted for lysosomal degradation are tagged by ubiquitin, which is recognised by endosomal ubiquitin receptors that select such cargo and engage membrane-deforming proteins to enact each transport step. Second, these endosomal ubiquitin receptors can themselves be ubiquitinated, resulting in their auto-inhibition and thus they are switched off. Third, many membrane-deforming proteins are also ubiquitinated, often resulting in their rapid and reversible inactivation. Their coordinated ubiquitination ensures that transport reactions within the endosomal pathway are processive and efficient.

Many of these ubiquitination events are driven by a family of ubiquitin ligases called Rsp5s, of which ITCH is a notable member. These ligases are located throughout the cell, so they must be closely controlled by 'adaptor' proteins to ensure that they ubiquitinate the correct targets at the right time and place. We propose that a protein called LITAF is the crucial adaptor that activates ITCH (and other Rsp5 ligases) upon the high-curvature membranes within the endosomal pathway and hence promotes the range of ubiquitination events that underpin the proper functioning of the endosomal pathway. We base our hypothesis on our findings: i) LITAF can itself support membrane curvature and is important for endosomal transport; ii) LITAF activates ITCH; iii) LITAF binds to endosomal ubiquitin receptors; iv) Depletion of LITAF and ITCH generate similar defects in membrane curvature within the endosomal pathway.

Our proposal has two aims. First, we seek to understand how LITAF activates ITCH, and hence behaves as a bona fide ITCH adaptor with the potential to activate ITCH on high-curvature membranes. Second, we will identify the proteins for which LITAF promotes ITCH-dependent ubiquitination. We believe that endosomal ubiquitin receptors may be a key class of such substrates, and we will test this. We will also perform a non-biased screen to look for other LITAF clients, looking out for important components that facilitate endosomal transport steps. For a small subset of these we will test the impact of their ubiquitination.

The endosomal pathway samples and modifies the complement of surface proteins and as such is vital to cell function. Membrane proteins are internalised and then sorted at the endosome towards lysosomal degradation or recycled to the surface, with each transport step coupling cargo recognition to changes in membrane curvature. The endosomal pathway is governed by ubiquitination. Cargo destined for lysosomes is ubiquitinated, and then sorted by ubiquitin receptors. In turn, ubiquitin receptors and other transport components are controlled by ubiquitination, often catalysed by the ITCH/Rsp5 family of ubiquitin ligases. What ensures that ITCH is activated in the vicinity of these substrates, upon high-curvature membranes within the endosomal pathway, is unclear.

We propose that LITAF, a monotopic integral membrane protein, acts as the primary ITCH/Rsp5 adaptor to facilitate such ubiquitination. We find that i) LITAF supports membrane curvature and promotes membrane transport steps that involve high membrane-curvature; ii) LITAF activates ITCH; iii) LITAF binds to endosomal ubiquitin receptors; iv) Depletion of LITAF and ITCH generate similar aberrant endosomal morphology, highlighting ITCH as the model Rsp5 ligase for dissecting LITAF function.

Aim 1. We will determine how LITAF activates ITCH, focusing on characteristics that LITAF shares with other ITCH/Rsp5 adaptors. LITAF appears oligomeric, which may be important both for promoting membrane curvature and activating several copies of ITCH co-ordinately. We will define the LITAF multimer, and then test if it recruits multiple copies of ITCH.
Aim 2. Endosomal ubiquitin receptors are one likely class of LITAF clients for ITCH-dependent ubiquitination. We will test if this is the case. To explore the broad range of LITAF clients we will screen which near-neighbours of LITAF are substrates for ubiquitination by ITCH and/or other Rsp5s, and for a subset of these test the impact of ubiquitination on their function.