Sensing Membrane Curvature in Macroautophagy

Abstract

In response to intracellular stress events ranging from starvation to pathogen invasion, the cell activates one or more forms of macroautophagy. The key event in these related pathways is the de novo formation of a new organelle called the autophagosome, which either surrounds and sequesters random portions of the cytoplasm or selectively targets individual intracellular challenges. Thus, the autophagosome is a flexible membrane platform with dimensions that ultimately depend upon the target cargo. The intermediate membrane, termed the phagophore or isolation membrane, is a cup-like structure with a clear concave face and a highly curved rim. The phagophore is largely devoid of integral membrane proteins; thus, its shape and size are governed by peripherally associated membrane proteins and possibly by the lipid composition of the membrane itself. Growth along the phagophore rim marks the progress of both organelle expansion and ultimately organelle closure around a particular cargo. These two properties, a reliance on peripheral membrane proteins and a structurally distinct membrane architecture, suggest that the ability to target or manipulate membrane curvature might be an essential activity of proteins functioning in this pathway. In this review, we discuss the extent to which membranes are naturally curved at each of the cellular sites believed to engage in autophagosome formation, review basic mechanisms used to sense this curvature, and then summarize the existing literature concerning which autophagy proteins are capable of curvature recognition.

Keywords: BAR domain; amphipathic helix; membrane curvature; reticulon domain.

Figure 1. Sources of membrane curvature associated with macroautophagy in the mammalian cell

Autophagosome biogenesis involves many structures that present strident positive curvature to the cytoplasm including very small vesicles, ER tubules, tubular protrusions from very small recycling endosome and golgi vesicles, and the rim of the expanding phagophore (see Table I). Proteins associated with each step in the organelle’s maturation have been shown to possess membrane curvature sensing in vitro and to rely on these motifs for proper function in vivo.

Figure 2. Phagophore dimensions

The phagophore or isolation membrane is a cup-shaped intermediate in autophagosome development. It includes a concave inner and convex outer surface as well as a highly curved rim running along the open edge of the cup. Labeled are the diameters of several autophagy-related membrane structures exhibiting high curvature. In addition, we note the typical distances between the bilayers in the growing isolation membrane or the mature autophagosome (lamellar spacing). Although this distance does not directly represent a curvature, it sets a kind of lower bound for the radius of curvature connecting the two bilayers at the rim. In practice, actual measures of rim curvature are mostly not available. We have surveyed a representative set of articles in the literature to generate the range of measures in Table I.

Figure 3. Mechanisms for proteins to recognize and interact with highly curved membranes

A) Peripheral proteins either recognize the shape of the membrane primarily through engagement with phospholipid headgroups or interrogate the hydrophobic core of the bilayer with membrane insertion sequences. (BAR domains often include both scaffolding and amphipathic helix motifs). B) Membrane insertion of poorly hydrophobic motifs, such as those commonly associated with curvature-sensing, relies on poor lipid packing in the bilayer to favor partitioning of protein motifs into the membrane. If we consider phospholipids as cylinders or cones, with phospholipid headgroups’ and acyl chains’ cross sectional areas defining the base ^, , we can see that on planar surfaces, the packing of conical lipid headgroups becomes less ideal. In order to minimize exposed hydrophobic surface area of the acyl chains, membranes can bend or inverted conical lipids or protein insertion motifs can sort to these areas of the bilayer.

Figure 4. Highly curved membranes allow efficient interaction and activation of autophagic machinery

Multiple protein sequence motifs, whether autophagy specific or not, have been shown to recognize structural deformation of target membranes. A common mechanism involves an alpha helix, consisting of hydrophobic residues flanked by hydrophilic residues, to penetrate the membrane. The distribution of these amino acids reflect the lipidic composition of the target membranes. For examples ALPS motifs were originally identified at the golgi (i.e. ArfGAP1) and are thought to be commonly associated membrane events early in the secretory cascade, while more charged amino acids are often involved in peripheral membrane targeting. In autophagy, proteins that propagate the nucleation, elongation, and completion stages of the autophagosome have each been described with curvature-dependent amphipathic helices including those that mimic ALPS motifs (human Atg14L) and those with more charged faces (human Atg3).