LAUSR

The concept of compact, ordered sphingolipid–cholesterol membrane domains that function as “rafts” with biologically relevant functions was first put forward 26 y ago (1). Since then, the field of “lipid rafts” has blossomed and matured in several thousand publications. It may therefore come as a bit of a surprise that the recent literature still carefully contemplates fundamental questions about lipid raft characteristics, roles, and even their very existence (2–4). The issue is not with the well-characterized existence of different lipid nanodomains and microdomains in cellular membranes (5, 6) but with nonunified ideas about what constitutes a lipid raft and a raft-dependent, biologically relevant function in living cells (2, 7). What if lipid rafts are not one thing but a host of different, short-lived nanodomains that dynamically and continuously change their lipid composition, protein interactions, and cell biological functions? In this issue of PNAS, Diaz-Rohrer et al., used live observation to characterize trafficking of membrane proteins to the plasma membrane (PM) depending on ordered versus disordered lipid association (8). The key observations are based on preparations of giant PM vesicles, membrane blebs derived from cultured cells that retain the lipid diversity, and protein content of living membranes (9). Using the same approach, the group had previously shown that protein palmitoylation, transmembrane domain (TMD) length, and sequence affect both raft association and PM localization (10). In the new study, the authors now propose that raft-mediated trafficking to the PM occurs via a raft-specific recycling pathway that has its checkpoint at the late endosome. Key to the study is the development of single TMD raft and nonraft fluorescently labeled probes that contain no known protein sequence affecting intracellular sorting (Fig. 1). Both raftand nonraft-associated probes trafficked to the PM through the secretory pathway; however, following constitutive endocytosis and arrival in Rab7-positive late endosomes, nonraft probes were sorted to be degraded in lysosomes, while raft probes were recycled back to the PM (Fig. 1). Based on experimental overexpression of guanosine diphosphate (GDP)or guanosine triphosphate (GTP)-locked Rab GTPases, this recycling process is indeed dependent on Rab7, but surprisingly not Rab11 or Rab4, the GTPases previously implicated in recycling back to the PM. Endomembrane trafficking has long been a prime target in the search for cell biological roles of lipid rafts (11). The study's authors have previously proposed a gradient of ordered membrane domains along the secretory and recycling pathways, where the PM is most enriched, endosomes intermediate, and lysosomes poor in ordered membranes (2) (Fig. 1). Sorting in the secretory and recycling pathways has been extensively studied and is regulated by a host of proteins, from vesicle coats to Rabs and other GTPases, that function in concert to regulate the dynamics of intracellular trafficking. Recycling endosomes are the best-characterized sorting stations for PM recycling, and raft-based sorting has been proposed already in the early days of lipid raft studies (12). However, early tests of the simplified “sorting to the ordered PM” idea with GPI-linked GFP and other raft probes already indicated that intracellular trafficking itineraries do not simply follow a gradient of ordered membrane and that additional sorting signals must be required (13). The late endosome has been investigated repeatedly as a hub for raft-associated protein sorting and shown to contain raft-type membrane domains of various compositions (14–16). The heterogeneity of membrane domains in compartments like