Conserved targeting information in mammalian and fungal peroxisomal tail-anchored proteins

The targeting signals and mechanisms of soluble peroxisomal proteins are well understood, whereas less is known about the signals and targeting routes of peroxisomal membrane proteins (PMP). Pex15 and PEX26, tail-anchored proteins in yeast and mammals, respectively, exert a similar cellular function in the recruitment of AAA peroxins at the peroxisomal membrane. But despite their common role, Pex15 and PEX26 are neither homologs nor they are known to follow similar targeting principles. Here we show that Pex15 targets to peroxisomes in mammalian cells, and PEX26 reaches peroxisomes when expressed in yeast cells. In both proteins C-terminal targeting information is sufficient for correct sorting to the peroxisomal membrane. In yeast, PEX26 follows the pathway that also ensures correct targeting of Pex15: PEX26 enters the endoplasmic reticulum (ER) in a GET-dependent and Pex19-independent manner. Like in yeast, PEX26 enters the ER in mammalian cells, however, independently of GET/TRC40. These data show that conserved targeting information is employed in yeast and higher eukaryotes during the biogenesis of peroxisomal tail-anchored proteins

Conserved targeting information in mammalian and fungal peroxisomal tail-anchored proteins

The targeting signals and mechanisms of soluble peroxisomal proteins are well understood, whereas less is known about the signals and targeting routes of peroxisomal membrane proteins (PMP). Pex15 and PEX26, tail-anchored proteins in yeast and mammals, respectively, exert a similar cellular function in the recruitment of AAA peroxins at the peroxisomal membrane. But despite their common role, Pex15 and PEX26 are neither homologs nor they are known to follow similar targeting principles. Here we show that Pex15 targets to peroxisomes in mammalian cells, and PEX26 reaches peroxisomes when expressed in yeast cells. In both proteins C-terminal targeting information is sufficient for correct sorting to the peroxisomal membrane. In yeast, PEX26 follows the pathway that also ensures correct targeting of Pex15: PEX26 enters the endoplasmic reticulum (ER) in a GET-dependent and Pex19-independent manner. Like in yeast, PEX26 enters the ER in mammalian cells, however, independently of GET/TRC40. These data show that conserved targeting information is employed in yeast and higher eukaryotes during the biogenesis of peroxisomal tail-anchored proteins

WRB and CAML are necessary and sufficient to mediate tail-anchored protein targeting to the ER membrane.

Tail-Anchored (TA) proteins are inserted into the endoplasmic reticulum (ER) membrane of yeast cells via the posttranslational Guided Entry of Tail-Anchored protein (GET) pathway. The key component of this targeting machinery is the ATPase Get3 that docks to the ER membrane by interacting with a receptor complex formed by the proteins Get1 and Get2. A conserved pathway is present in higher eukaryotes and is mediated by TRC40, homolog of Get3, and the recently identified membrane receptors WRB and CAML. Here, we used yeast lacking the GET1 and GET2 genes and substituted them with WRB and CAML. This rescued the growth phenotypes of the GET receptor mutant. We demonstrate that WRB and CAML efficiently recruit Get3 to the ER membrane and promote the targeting of the TA proteins in vivo. Our results show that the membrane spanning segments of CAML are essential to create a functional receptor with WRB and to ensure TA protein membrane insertion. Finally, we determined the binding parameters of TRC40 to the WRB/CAML receptor. We conclude that together, WRB and CAML are not only necessary but also sufficient to create a functional membrane receptor complex for TRC40. The yeast complementation assay can be used to further dissect the structure-function relationship of the WRB/CAML heteromultimer in the absence of endogenous receptor proteins

The transmembrane domains of CAML are essential for a functional WRB/CAML receptor complex.

<p>(A) Schematic representation of CAML-Get2 chimeras. Position of transmembrane domains (TMD) are indicated. (B) <i>get1/get2</i> yeast cells carrying a genomically GFP-tagged version of Get3 were transformed with a plasmid containing the coding sequence of WRB in combination with CAML or CAML-Get2 chimeras. Subcellular Get3-GFP localization was analyzed by fluorescence microscopy. (C) <i>get1/get2</i> yeast cells were transformed with a plasmid containing the coding sequence of GFP-tagged Sed5 and Get1/Get2 or WRB in combination with CAML or CAML-Get2 chimeras. Subcellular GFP-Sed5 localization was analyzed by fluorescence microscopy. (D) Images taken in (C) were quantified to determine the distribution of fluorescence across bins of different pixel intensity for each strain. A minimum of 42 cells was analyzed per strain.</p

In combination, WRB and CAML rescue Get3 localization at the ER membrane and TA protein targeting.

<p>(A) <i>get1/get2</i> yeast cells carrying a genomically GFP-tagged version of Get3 were transformed with combinations of WRB, CAML, Get1 and Get2 encoding constructs. Subcellular Get3-GFP localization was analyzed by fluorescence microscopy. (B) <i>get1/get2</i> yeast cells were transformed with a plasmid containing the coding sequence of GFP-tagged Sed5 and combinations of WRB, CAML, Get1 and Get2 encoding constructs. Subcellular GFP-Sed5 localization was analyzed by fluorescence microscopy. (C) Images taken in (B) were quantified to determine the distribution of fluorescence across bins of different pixel intensity for each strain. A minimum of 41 cells was analyzed per strain.</p

Reflectometric Interference Spectroscopy (RIfS) and Surface plasmon resonance (SPR) measurements.

<p>Affinity constants of TRC40 and TRC40/RAMP4 complex for rough microsomes, WRBcc and CAMLcyt. Values represent the average calculated from three independent measurements.</p

WRB and CAML rescue the growth phenotypes of <i>get1/get2</i> yeast cells.

<p><i>get1/get2</i> yeast cells were transformed with combinations of WRB, CAML, Get1 and Get2 encoding constructs and serial dilutions spotted on different conditions: HC plates incubated at 30°C (control), 37°C+CuSO₄, 39°C, H₂O₂, hydroxyurea, tunicamycin, hygromycin.</p

Summary of the results obtained in this study.

<p>¹ growth rescue at 39°C.</p><p>‘n.a.’ = not assayed’.</p

The Protein Targeting Factor Get3 Functions as ATP-Independent Chaperone under Oxidative Stress Conditions

Exposure of cells to reactive oxygen species (ROS) causes a rapid and significant drop in intracellular ATP levels. This energy depletion negatively affects ATP-dependent chaperone systems, making ROS-mediated protein unfolding and aggregation a potentially very challenging problem. Here we show that Get3, a protein involved in ATP-dependent targeting of tail-anchored (TA) proteins under nonstress conditions, turns into an effective ATP-independent chaperone when oxidized. Activation of Get3's chaperone function, which is a fully reversible process, involves disulfide bond formation, metal release, and its conversion into distinct, higher oligomeric structures. Mutational studies demonstrate that the chaperone activity of Get3 is functionally distinct from and likely mutually exclusive with its targeting function, and responsible for the oxidative stress-sensitive phenotype that has long been noted for yeast cells lacking functional Get3. These results provide convincing evidence that Get3 functions as a redox-regulated chaperone, effectively protecting eukaryotic cells against oxidative protein damage