Predicting the targeting of tail-anchored proteins to subcellular compartments in mammalian cells

Abstract

Tail-anchored (TA) proteins contain a single transmembrane domain (TMD) at the C-terminus that anchors them to the membranes of organelles where they mediate critical cellular processes. Accordingly, mutations in genes encoding TA proteins have been identified in a number of severe inherited disorders. Despite the importance of correctly targeting a TA protein to its appropriate membrane, the mechanisms and signals involved are not fully understood. In this study, we identify additional peroxisomal TA proteins, discover more proteins that are present on multiple organelles, and reveal that a combination of TMD hydrophobicity and tail charge determines targeting to distinct organelle locations in mammals. Specifically, an increase in tail charge can override a hydrophobic TMD signal and re-direct a protein from the ER to peroxisomes or mitochondria and vice versa. We show that subtle changes in those parameters can shift TA proteins between organelles, explaining why peroxisomes and mitochondria have many of the same TA proteins. This enabled us to associate characteristic physicochemical parameters in TA proteins with particular organelle groups. Using this classification allowed successful prediction of the location of uncharacterized TA proteins for the first time.

A role for Mitochondrial Rho GTPase 1 (MIRO1) in motility and membrane dynamics of peroxisomes

“…Additionally, in a high‐throughput interaction study, MIRO1 was identified as a PEX19 interaction partner 37. These findings are also consistent with the known organelle targeting signals: MIRO1 possesses a transmembrane domain (TMD) with relatively low hydrophobicity (GRAVY, 1.3) and a moderate net charge in the tail region (1.9), which based on our previous work would be indicative of a TA protein that localises predominantly to mitochondria but has a potential for peroxisomal targeting 34. Overall, our findings support a dual localisation of MIRO1 at mitochondria and peroxisomes.…”

“…As MIRO1 can alter peroxisome distribution and motility, it is likely one of the yet unidentified adaptors for microtubule‐based peroxisome motility in mammalian cells. This assumption is further supported by recent findings showing that mitochondria and peroxisomes share many TA membrane proteins and their functions due to their close cooperation and co‐evolution in mammalian cells 34. During the submission of our work, Okumoto et al 55 revealed that distinct MIRO1 splice variants show different targeting to mitochondria and peroxisomes in HEK cells, with MIRO1‐variant 4 being more specific for peroxisomes.…”

“…The targeting of all known TA proteins to peroxisomes requires the peroxisomal import receptor/chaperone PEX19 34. For MIRO1, PEX19 binding was shown by immunoprecipitation after co‐expression of Myc‐MIRO1 and HA‐PEX19 in COS‐7 cells (Figure 1B) suggesting a role for PEX19 in the targeting of MIRO1 to peroxisomes.…”

Miro1 – the missing link to peroxisome motility

“…Interestingly, numerous TA proteins are dually targeted to both peroxisomes and mitochondria [23]. This targeting appears to be largely dependent on the biochemical properties of their C-terminal amino acid sequence (principally the hydrophobicity of the TMD and the net charge in the tail region) and, as such, it can be characterized and used to predict the targeting of TA proteins [12]. Interestingly, Miro1 has an identical TMD hydrophobicity and tail charge to that of Mff, another dually targeted protein involved in mitochondrial and peroxisomal division [24].…”

Peroxisomal ACBD4 interacts with VAPB and promotes ER-peroxisome associations

“…These characteristic properties dictate that TA proteins are post-translationally sorted to their target membrane with the N-terminus facing the cytosol 23 . In another recent study we investigated the targeting properties of TA proteins, discovering the importance of interplay between TMD hydrophobicity and tail-charge, and developed a statistical model to predict cellular localization of TA proteins based on these physicochemical parameters 24 . Using this bioinformatics prediction tool we identified an isoform of ACBD4 (isoform 2), a predicted TA protein of unknown function and localization, as a potential peroxisomal protein and confirmed this by expression of Myc-ACBD4iso2 in COS-7 cells 24 .…”