Recent advances have uncovered the general protein apparatus used by all eukaryotes for intracellular transport, including secretion and endocytosis, and for triggered exocytosis of hormones and neurotransmitters. Membranes are shaped into vesicles by cytoplasmic coats which then dissociate upon GTP hydrolysis. Both vesicles and their acceptor membranes carry targeting proteins which interact specifically to initiate docking. A general apparatus then assembles at the docking site and fuses the vesicle with its target.

Mechanisms of intracellular protein transport

Sorting of transmembrane proteins to endosomes and lysosomes is mediated by signals present within the cytosolic domains of the proteins. Most signals consist of short, linear sequences of amino acid residues. Some signals are referred to as tyrosine-based sorting signals and conform to the NPXY or YXXO consensus motifs. Other signals known as dileucine-based signals fit [DE]XXXL[LI] or DXXLL consensus motifs. All of these signals are recognized by components of protein coats peripherally associated with the cytosolic face of membranes. YXXO and [DE]XXXL[LI] signals are recognized with characteristic fine specificity by the adaptor protein (AP) complexes AP-1, AP-2, AP-3, and AP-4, whereas DXXLL signals are recognized by another family of adaptors known as GGAs. Several proteins, including clathrin, AP-2, and Dab2, have been proposed to function as recognition proteins for NPXY signals. YXXO and DXXLL signals bind in an extended conformation to the mu2 subunit of AP-2 and the VHS domain of the GGAs, respectively. Phosphorylation events regulate signal recognition. In addition to peptide motifs, ubiquitination of cytosolic lysine residues also serves as a signal for sorting at various stages of the endosomal-lysosomal system. Conjugated ubiquitin is recognized by UIM, UBA, or UBC domains present within many components of the internalization and lysosomal targeting machinery. This complex array of signals and recognition proteins ensures the dynamic but accurate distribution of transmembrane proteins to different compartments of the endosomal-lysosomal system.

Signals for Sorting of Transmembrane Proteins to Endosomes and Lysosomes

Mechanisms of intracellular protein transport.

THE definition of cellular organelles has evolved over the last hundred years largely driven by morphologic observations, but more recently has been supplemented and complemented by functional and biochemical studies (Palade, 1975) . Thus, organelles are now identified both by their morphology and by the set ofcomponents that comprise them . Determining how organelle identity is established and maintained and how newly synthesized protein and membrane are sorted to different organelles are the central issues of organellogenesis . Essential to the many cellular functions that take place within the central vacuolar system (which consists ofthe ER, Golgi apparatus, secretory vesicles, endosomes, and lysosomes) is membrane traffic which mediates the exchange of components between different organelles . There are two critical characteristics of membrane traffic . First, only certain sets oforganelles exchange membrane and the patterns of this exchange define what are called membrane pathways . Second, multiple pathways intersect at specific points within the central vacuolar system . For specific components to "choose" the correct pathway at such points of crossing, mechanisms exist to impose choices on specific molecules . This process is called sorting . The characteristicsofeachorganelle within the central vacuolar system are likely to be intimately tied to the properties ofmembrane traffic . An imbalance in the magnitude ofmembrane input into and egress from an organelle would have profound effects on the size ofthat compartment . In addition, failures in sorting or aberrations in targeting pathways would be expected to profoundly affect the identity of individual organelles . Recently, the relationship between the control of membrane traffic and the maintenance of organelle structure has been investigated with the use ofa remarkable drug, brefeldin A (BFA).' In this review we will summarize recent findings with BFA and propose some speculative models concerning the mechanism and regulation ofmembrane traffic within the central vacuolar system .

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Brefeldin A: insights into the control of membrane traffic and organelle structure.

Endocytosis in eukaryotic cells is characterized by the continuous and regulated formation of prolific numbers of membrane vesicles at the plasma membrane. These vesicles come in several different varieties, ranging from the actin-dependent formation of phagosomes involved in particle uptake, to smaller clathrin-coated vesicles responsible for the internalization of extracellular fluid and receptor-bound ligands. In general, each of these vesicle types results in the delivery of their contents to lysosomes for degradation. The membrane components of endocytic vesicles, on the other hand, are subject to a series of highly complex and iterative molecular sorting events resulting in their targeting to specific destinations. In recent years, much has been learned about the function of the endocytic pathway and the mechanisms responsible for the molecular sorting of proteins and lipids. This review attempts to integrate these new concepts with long-established views of endocytosis to present a more coherent picture of how the endocytic pathway is organized and how the intracellular transport of internalized membrane components is controlled. Of particular importance are emerging concepts concerning the protein-based signals responsible for molecular sorting and the cytosolic complexes responsible for the decoding of these signals.

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Endocytosis and molecular sorting

CONTENTS INTRODUCTION 15 1 COAT COMPONENTS 153 Clathrin Triskelions and Adaptors ....... 153 Function and Distribution of Adaptors 156 COATED PITS AS MOLECULAR FILTERS ......... ... .. 159 Clathrin-Mediated Endocytosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 Clathrin-Mediated Intracellular Sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 62 Defective Sorting in Yeast Cells Lacking Clathrin ... 165 Interaction of Receptors with Adaptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166 CONCLUSIONS AND PROBLEMS 167

Clathrin, Adaptors, and Sorting

A family of coated vesicle proteins, with molecular weights of- 100,000 and designated 100K, has been implicated in both coat assembly and the attachment of clathrin to the vesicle membrane. These proteins were purified from extracts of bovine brain coated vesicles by gel filtration, hydroxylapatite chro- matography, and preparative SDS PAGE. Peptide mapping by limited proteolysis indicated that the polypeptides making up the three major 100K bands have distinct amino acid sequences. When four rats were immunized with total 100K protein, each rat responded differently to the different bands, although all four antisera cross-reacted with the 100K proteins of human placental coated vesicles. After affinity puri- fication, two of the antisera were able to detect a 100K band on blots of whole 3T3 cell protein and were used for immunofluorescence, double labeling the cells with either rabbit anti-clathrin or with wheat germ lectin as a Golgi apparatus marker. Both anti- sera gave staining that was coincident with anti-clath- fin, with punctate labeling of the plasma membrane and perinuclear Golgi apparatus labeling. Thus, the 100K proteins are present on endocytic as well as Golgi-derived coated pits and vesicles. The punctate patterns were nearly identical with anti-100K and anti-clathrin, indicating that when vesicles become uncoated, the 100K proteins are removed as well as clathrin. One of the two antisera gave stronger plasma membrane labeling than Golgi apparatus labeling when compared with the anti-clathrin antiserum. The other antiserum gave stronger Golgi apparatus label- ing. Although we have as yet no evidence that these two antisera label different proteins on blots of 3T3 cells, they do show differences on blots of bovine brain 100K proteins. This result, although prelimi- nary, raises the possibility that different 100K proteins may be associated with different pathways of mem- brane traffic.

Margaret S. Robinson

Papers

Clathrin, Adaptors, and Sorting

Immunofluorescent of 100K Coated Vesicle Proteins