All about that fat: Lipid modification of proteins in Cryptococcus neoformans

Abstract

Lipid modification of proteins is a widespread, essential process whereby fatty acids, cholesterol, isoprenoids, phospholipids, or glycosylphospholipids are attached to polypeptides. These hydrophobic groups may affect protein structure, function, localization, and/or stability; as a consequence such modifications play critical regulatory roles in cellular systems. Recent advances in chemical biology and proteomics have allowed the profiling of modified proteins, enabling dissection of the functional consequences of lipid addition. The enzymes that mediate lipid modification are specific for both the lipid and protein substrates, and are conserved from fungi to humans. In this article we review these enzymes, their substrates, and the processes involved in eukaryotic lipid modification of proteins. We further focus on its occurrence in the fungal pathogen Cryptococcus neoformans, highlighting unique features that are both relevant for the biology of the organism and potentially important in the search for new therapies.

Keywords: Cryptococcus; GPI-anchored proteins; isoprenylation; lipid modification; myristoylation; palmitoylation; prenylation; protein lipidation.

Fig. 1. GPI-anchored proteins in fungi

(A) Chemical structure of a GPI core, with the two acyl chains (x and y, which may differ in length and degree of saturation) of the phosphatidylinositol (PI) embedded in the membrane. The inositol phospholipids are most commonly diacyl-PI, but may also be lysoacyl-, alkylacyl-, or alkenylacyl-PI or inositolphosphoceramide. In fungi (as well as mammals), the C2 position of the inositol ring is often acylated (denoted by the asterisk), but in C. neoformans this substitution is mostly palmitate. (B) After anchor addition (and potential palmitoylation) in the ER lumen (pink), GPI-proteins enter the secretory pathway where they are further modified and trafficked in secretory vesicles to the PM (i); fusion of these vesicles with the PM exposes the GPI-protein cargo on the extracellular surface. Once at the cell surface, GPI-proteins can segregate into lipid rafts (purple phospholipids; ii) or, alternatively, can be translocated with part of their anchor (cleaved between glucosamine and mannose) to covalent linkage with cell wall (CW) β-1,6-glucan polymers (green tubes; iii). PM- or CW-localized GPI-proteins can be released into the extracellular space by the action of PI-specific phospholipases (PI-PLC; iv) or CW β-glucanases (v). The CW is a complex structure that surrounds the entire cell (Doering, 2009), but for clarity it is depicted only at the right of the cartoon and only the β-glucan fibers are shown.

Fig. 2. Palmitoylation acts as a cue for traffic and sorting towards specific membranes

(A) Palmitoylation of integral membrane proteins in the ER or Golgi directs them to the PM (right). If this does not occur, the proteins are retained in the ER, where they aggregate (left). (B) Palmitoylation may also direct PM transmembrane proteins into lipid rafts (purple phospholipids; right). For these proteins, the absence of palmitoylation results in mislocalization. In a subset of palmitoylated proteins, the modified Cys is near a Lys that is targeted for ubiquitin (Ub) addition; palmitoylation of the Cys prevents this from occurring. In the absence of lipidation the Ub is added, targeting the protein for lysosomal degradation (left).

Fig. 3. Consequences of myristoylation on protein function

(A) The Arf GTPase cycle illustrates myristoylation-dependent membrane recruitment. In the inactive GDP-state, the myristoyl moiety (black zigzag) is sequestered in a hydrophobic pocket of the protein while the polypeptide samples membranes via its amphipathic helix (multicolored cylinder). GEF (GDP-exchange factor) binding transiently stabilizes Arf (blue) at the membrane and mediates GTP loading; this displaces GDP, leading to closure of the pocket and exclusion of the myristate. The insertion of the excluded myristate into the lipid bilayer leads to stable membrane association of the activated Arf (green), which can then recruit effectors and regulate membrane trafficking (orange arrow). Subsequent inactivation of Arf, catalyzed by its GAP (GTPase activating protein), reverses the process, releasing Arf from the membranes to restart the cycle. (B) Lipidation is critical for G-alpha protein function. While unmodified G-alpha (blue) is cytosolic, myristoylation of this protein mediates its transient membrane retention at the ER as it moves into the Golgi (i). This makes the protein accessible to a Golgi-localized PAT, which adds a second lipid modification (ii). This combination firmly anchors the protein to membranes and provides a signal for trafficking to the PM. There the dually-modified alpha subunit interacts with its partners, the G-βγ heterodimer (itself typically prenylated and sometimes additionally palmitoylated) and a 7-transmembrane domain sensory receptor, forming a signaling-competent unit: the G protein coupled receptor (GPCR; (iii)).

Fig. 4. Lipidation directs Ras localization and function

(i) FTase farnesylates cytosolic Ras proteins (lower right). This mediates their association with the ER membrane where further processing of the CAAX box occurs. Farnesylated Ras then traffics to the Golgi (lower left) where it is palmitoylated by a Golgi-associated PAT (ii) and the dually modified protein (dark blue) proceeds to the PM via the secretory pathway. At the PM Ras can be activated by guanine nucleotide exchange factors (GEFs; (iii)); the active GTP-bound form (green) functions as a signaling molecule (orange arrow) until it is inactivated by GTPase activating proteins (GAPs; (iv)). PM-localized Ras may also be depalmitoylated by a PPT (v), leading to its dissociation from the PM. It then diffuses and samples various cellular membranes, rejoining the cycle at the Golgi where its membrane localization is stabilized by palmitate addition.