Targeting of tail‐anchored proteins to Trichomonas vaginalis hydrogenosomes

Abstract

Summary Tail‐anchored (TA) proteins are membrane proteins that are found in all domains of life. They consist of an N‐terminal domain that performs various functions and a single transmembrane domain (TMD) near the C‐terminus. In eukaryotes, TA proteins are targeted to the membranes of mitochondria, the endoplasmic reticulum (ER), peroxisomes and in plants, chloroplasts. The targeting of these proteins to their specific destinations correlates with the properties of the C‐terminal domain, mainly the TMD hydrophobicity and the net charge of the flanking regions. Trichomonas vaginalis is a human parasite that has adapted to oxygen‐poor environment. This adaptation is reflected by the presence of highly modified mitochondria (hydrogenosomes) and the absence of peroxisomes. The proteome of hydrogenosomes is considerably reduced; however, our bioinformatic analysis predicted 120 putative hydrogenosomal TA proteins. Seven proteins were selected to prove their localization. The elimination of the net positive charge in the C‐tail of the hydrogenosomal TA4 protein resulted in its dual localization to hydrogenosomes and the ER, causing changes in ER morphology. Domain mutation and swap experiments with hydrogenosomal (TA4) and ER (TAPDI) proteins indicated that the general principles for specific targeting are conserved across eukaryotic lineages, including T. vaginalis; however, there are also significant lineage‐specific differences.

Hydrogenosomal tail-anchored proteins are targeted to both mitochondria and ER upon their expression in yeast cells

“…Additionally, we observed a large amount of the GFP signal in the vacuole, suggesting that a subpopulation of the protein was degraded in the vacuole. We concluded that, in yeast cells, HA-TA4 is mainly localized to the ER and only partially to mitochondria, contrary to the situation in T. vaginalis where it was found exclusively in hydrogenosomes [8]. This implies that the targeting signals and their decoding are not completely conserved between T. vaginalis and S. cerevisiae.…”

“…The obtained DNA fragments were inserted downstream of the 3xHA tag into the yeast expression vector pYX142, digested with the same restriction enzymes. To construct the pYX142-3HA-TA7 plasmid, the coding sequence for TA7 was amplified by PCR from pTagVag-2HA-TA7 [ 8 ], using primers containing the restriction sites for EcoRI and NheI. This DNA fragment was inserted downstream of the 3xHA tag of the plasmid pRS316- FIS1 pr-3HA-Fis1(cyt)- FIS1ter [ 15 ], which was digested with the same restriction enzymes.…”

“…The cDNAs of TA4 and TA10 were amplified by PCR with primers containing NcoI and SalI restriction sites, using pVagTag-2HA-TA4 and pVagTag-2HA-TA10 [8], respectively, as templates. The obtained DNA fragments were inserted downstream of the 3xHA tag into the yeast expression vector pYX142, digested with the same restriction enzymes.…”

“…Recently, several putative tail-anchored proteins residing in the hydrogenosomes of T. vaginalis were identified [8]. To study the conservation of the targeting of TA proteins between T. vaginalis and S. cerevisiae, we expressed in yeast cells out of the five newly identified hydrogenosomal proteins the three proteins that have less hydrophobic TMS, a typical characteristic of mitochondrial TA proteins, and investigated their localization and topology.…”

“…Proteomic analysis identified 70 putative hydrogenosomal membrane proteins in T. vaginalis, including 12 putative tail-anchored (TA) proteins [7,8]. TA proteins are single span membrane proteins with a transmembrane segment (TMS) localized close to the C-terminus of the polypeptide.…”