MUBs, a Family of Ubiquitin-fold Proteins That Are Plasma Membrane-anchored by Prenylation

Abstract

Ubiquitin (Ub)-fold proteins are rapidly emerging as an important class of eukaryotic modifiers, which often exert their influence by post-translational addition to other intracellular proteins. Despite assuming a common ␤-grasp threedimensional structure, their functions are highly diverse because of distinct surface features and targets and include tagging proteins for selective breakdown, nuclear import, autophagic recycling, vesicular trafficking, polarized morphogenesis, and the stress response. Here we describe a novel family of Membrane-anchored Ub-fold (MUB) proteins that are present in animals, filamentous fungi, and plants. Extending from the C terminus of the Ub-fold is typically a cysteinecontaining CAAX (where A indicates aliphatic amino acid) sequence that can direct the attachment of either a 15-carbon farnesyl or a 20-carbon geranylgeranyl moiety in vitro. Modified forms of several MUBs were detected in transgenic Arabidopsis thaliana, suggesting that these MUBs are prenylated in vivo. Both cell fractionation and confocal microscopic analyses of Arabidopsis plants expressing GFP-MUB fusions showed that the modified forms are membrane-anchored with a significant enrichment on the plasma membrane. This plasma membrane location was blocked in vivo in prenyltransferase mutants and by mevinolin, which inhibits the synthesis of prenyl groups. In addition to the five MUBs with CAAX boxes, Arabidopsis has one MUB variant with a cysteine-rich C terminus distinct from the CAAX box that is also membrane-anchored, possibly through the attachment of a long chain acyl group. Although the physiological role(s) of MUBs remain unknown, the discovery of these prenylated forms further expands the diversity and potential functions of Ub-fold proteins in eukaryotic biology.The superfamily of ubiquitin (Ub) 4 -type proteins has emerged over the last decade as an influential set of post-translational modifiers in eukaryotes. These small proteins (ϳ75-180 amino acids) contain a signature ␤-grasp (or Ub-fold) core domain, created by an ␣-helix transversing a groove created by an arched four-strand ␤-sheet (1). Extending from the core is a short flexible C-terminal extension that enables their attachment to other molecules through the terminal carboxyl group. The first and best-known member of this family is Ub (2, 3). It functions by becoming ligated to other proteins through an isopeptide bond between the C-terminal glycine of Ub and free lysine ⑀-amino groups in the target. The main role of Ub is to direct selective proteolysis by assembling chains of Ub monomers linked internally through Lys-48 onto various target proteins to promote their recognition by the 26 S proteasome. Others include roles in DNA repair via attachment of Lys-63-linked chains and endomembrane trafficking, transcription, and chromatin remodeling via attachment of Ub monomers (2, 3).Since the discovery of Ub, a number of related modifiers have been found, including Related to Ub-1 (RUB1 or NEDD8), Small-Ub-like Modifier (SUMO), Autophagy (ATG)-8 an...

Arabidopsis Membrane-anchored Ubiquitin-fold (MUB) Proteins Localize a Specific Subset of Ubiquitin-conjugating (E2) Enzymes to the Plasma Membrane

“…AtMUB3 and AtMUB4 are representative of the two prenylation modifications: geranylgeranylation and farnesylation, respectively (26). The AtMUB3/4 subgroup also represents the residue variation seen in the Ub Ile 44 -homologous surface, where a number of UBDs are known to interact (Fig.…”

“…To create Gateway entry vectors, AtMUB3 and AtMUB4 were PCR-amplified from published pET28 vectors (26) with forward primers including a 5Ј-CACC sequence and reverse primers designating 3Ј-CAAX or 3Ј-SAAX; products were directionally cloned into pENTR/D-TOPO (Invitrogen). AtCOP10 and AtUEV1B were amplified from Columbia-0 cDNA and HsMUB from SK-HEP cDNA (38) and similarly cloned into pENTR/D-TOPO.…”

“…The six MUB genes of Arabidopsis are divided by sequence homology into three subgroups, MUB1/2, MUB3/4, and MUB5/6, suggesting functional diversification of plant MUBs. In contrast, multicellular fungi and animals have only a single MUB gene, and no MUB gene is evident in yeast (26). Although the structure and posttranslational prenyl modifications of MUBs have been well characterized, the function of these Ub-fold proteins is unknown.…”

“…In planta, AtMUB membrane localization is prenylation-dependent, where mutation of the prenyl attachment CAAX cysteine to a serine (SAAX) prevents processing and causes accumulation of non-membrane-localized MUB. Likewise, Arabidopsis prenyltransferase mutants block membrane localization of endogenous AtMUB1 (26). The six MUB genes of Arabidopsis are divided by sequence homology into three subgroups, MUB1/2, MUB3/4, and MUB5/6, suggesting functional diversification of plant MUBs.…”

“…Prenylation and membrane localization preclude attachment of MUBs to target proteins. MUBs from nematodes, insects, fish, and mammals, including humans, are prenylated in vitro (26). In planta, AtMUB membrane localization is prenylation-dependent, where mutation of the prenyl attachment CAAX cysteine to a serine (SAAX) prevents processing and causes accumulation of non-membrane-localized MUB.…”