Sorting and traffic in the central vacuolar system

Cell, Vol. 57, 703-706, June 2, 1989, Copyright 0 1989 by Cell Press

M inireview

Sorting and Traffic in the Central Vacuolar System Richard D. Klausner Cell Biology and Metabolism Branch National Institute of Child Health and Human Development National Institutes of Health Bethesda, Maryland 20892

Regulation of intracellular traffic is one of the central issues in cell biology. In particular, the processes by which intracellular organelles are formed and maintained have received a tremendous amount of attention over the last few years. The protein components that both characterize and distribute among the various intracellular membranebounded organelles all begin their lives in the cytosol. Specific targeting signals within the protein are responsible for co- or posttranslational dispersal of proteins to their appropriate target organelles. Each independent organelle system in the cell appears to have a unique site of entry for proteins from the cytosol. Targeting sequences have been defined in proteins destined for the endoplasmic reticulum (ER), mitochondria, chloroplasts, nuclei, and peroxisomes. The ER serves as a port of entry from which lumenal and membrane proteins can be targeted through a complex organellar system. The organelles within this central vacuolar system include the rough and smooth ER, the cis-, medial, and rrans-Golgi, the transQolgi network (TGN), secretory vesicles, secretory granules, the endosomal system, lysosomes, and the plasma membrane (see figure). In addition, an unknown variety of transitional and/or transport organelles mediate the communication between these multiple compartments. Each of these organelles possesses characteristic resident membrane proteins that define the unique and differentiated structure and function of that compartment. Although the trafficking and sorting of proteins through this central organellar system have been the focus of intense interest since Palade (1975) and co-workers formulated the general outline of the secretory pathway, we are still far from a molecular description of these events. This minireview discusses briefly some of the current models that attempt to describe the principles and mechanisms of trafficking within the central vacuolar system. The Bulk Flow and Retention Model: A One-Choice System Two sets of experiments set the stage for the formulation of the bulk flow/retention hypothesis. Metaphorically stated, this model envisions the central vacuolar system as a flowing river, beginning in the ER and emptying into the outside world at the plasma membrane. Each defined organelle sits in a set order, defining segments of this river. All components of and within the organellar system are swept along in this flow. The resident proteins that define each organelle possess a unique signal that retains them at their appropriate site, preventing them from continuing on. Thus at every organelle along this river, only one

choice is made for any component: either stop (retention) or continue on. The simplicity of this model makes it attractive. Munro and Pelham (1987) first described a retention signal that appeared responsible for keeping proteins within the ER. This retention signal was defined as the tetrapeptide Lys-Asp-Glu-Leu (KDEL), which was found at the carboxy1 termini of several soluble proteins that remain within the ER. The ability to transfer retention by the addition of this tetrapeptide to the carboxyl terminus of an otherwise secreted protein strongly supported the proposed role of this sequence as a retention signal. Does this observation imply that retention establishes the identity of the organelles within the secretory pathway? KDEL-mediated retention so far appears to be only applicable to soluble, lumenal proteins. Although the Iumenal components of an organelle (e.g., lysosomes) may help to define it, it is most likely integral membrane proteins that establish organelle identity. Thus we must know whether the finding of a retention signal for soluble proteins tells us that retention signals determine the residency of membrane proteins. If we accept that movement through the organellar system is always through membrane-bounded structures, then it seems reasonable that soluble proteins could avoid completely traversing the secretory pathway only by interacting with (being retained by) an integral membrane receptor. By analogy, all components in the endocytic system that are free in the fluid phase share the fate of reaching lysosomes relatively efficiently. However, transferrin, which enters the same endocytic vesicles, follows a different fate and is recycled to the cell surface. This difference

.-

I on

0

Y3XEKXY GRANULE

ENWPLASMIC AETICUUJM

Cell 704

is explainable by the fact that transferrin is retained on its receptor and therefore accompanies the targeted movement of a membrane protein, whereas most ligands are released within the endosome from their receptors (Goldstein et al., 1985). Whatever the role of the KDEL sequence for soluble proteins, at this time there is no evidence for specific retention signals to explain the residency of the normal integral membrane protein components of any of the organelles of the vacuolar secretory system, although there is some evidence for retention within the ER of viral integral membrane proteins (see below). The results of a study by Rothman and colleagues (Wieland et al., 1987) led them to conclude that there is an absence of specific signals for the selective transport of proteins through the secretory system, leaving the presence of retention signals as the only option apparently available. These investigators measured the apparent rate of bulk flow from the ER through the secretory pathway to the outside of the cell by the use of a cell-permeable tripeptide that could serve as an acceptor for N-linked glycosylation. The N-linked glycosylation was assumed to take place within the lumen of the ER, and this modification was responsible for trapping the tripeptide in the fluid phase of the ER for transport through the secretory pathway. There was a lag time of only 5-10 min to release glycosylated peptide from the cell, which is faster than the secretion rate of any known newly synthesized membrane protein or secreted protein. The authors concluded that there could not be specific transport signals because the existence of such signals would lead to the prediction of transport rates equal to or faster than nonselective fluidphase movement. At least two assumptions are required to accept the validity of Wieland et al.‘s conclusion. The first is that glycosylation and release from the cells of their tripeptide indeed represent entrapment within the ER and transport as a fluid-phase marker through the normal biosynthetic pathway. The second is that newly synthesized proteins that have entered the ER contain any potential transport signal at the time of their synthesis. It may be that attaining a functional transport signal, either due to conformational maturation, assembly, or posttranslational modification, is the rate-limiting step in transport. Any such maturation would impose an obligatory lag period on this first step of transport. Thus, a comparison between the bulk flow rates of an inert, modified tripeptide and the secretion rates of newly synthesized proteins may not be an appropriate basis for distinguishing the types of signals that underly the movement of those proteins through the vacuolar system. Moving Through the System: Signals and Requirements In principle, two types of information could be employed to determine the fate of proteins within the organellar system: information to retain a protein in a given organelle and information to transport a protein out of a particular organelle. However, it is not that easy to determine which of these is operating. The only good evidence for retention is to detect physical association of a protein with another resident component of an organelle, and to correlate this

association with the protein’s localization. In other words, retention implies the existence of a “receptor” responsible, by binding, for determining the absence of movement of its target protein. Analogously, transport information might be established by detecting a specific association of a protein with a component of a transport vesicle, but there could be other types of transport information utilized. Criteria other than detecting a transport or retention “receptor” can be derived from genetic engineering of membrane proteins. For example, if removing a particular region of a protein releases retention and this region can transfer retention to heterologous proteins, a retention signal can be proposed. Retention in, versus transport out of, the ER has been the object of much study over the last few years (Pfeffer and Rothman, 1987). Most work addressing retention versus transport signals has focused on the exiting of proteins from the ER. Evidence for retention has been derived from studies in which certain mutant viral glycoproteins cannot get out of the ER because they fail to oligomerize normally in that organelle (Rose and Doms, 1988). These unassembled membrane proteins are associated with another resident component of the ER, the immunoglobulin heavy chain binding protein (BiP), and it is this interaction that has been hypothesized to retain the protein within the ER. However, mutations in the cytoplasmic tails of these same viral glycoproteins that neither prevent oligomerization nor apparently result in association with BiP also result in failure of the proteins to leave the ER. Do these mutations reflect an undetectable interaction responsible for retention or the failure to attain competence for transport? Another viral transmembrane protein, the adenovirus El9 protein, is retained within the ER. Studies have demonstrated that a region of the cytoplasmic tail of this protein might be responsible for retention (Paabo et al., 1988) since removal of a portion of the tail resulted in transport of the protein out of the ER. Once again, however, we can ask whether these mutations destroy a specific retention interaction or whether they remove a constraint to attaining transport competence. While there is no direct evidence for retention signals in resident membrane proteins of any organelle, there is a precedent for a signal required for transport. Thus in the only well-studied system in which proteins selectively enter a transport vesicle-the coated pits and vesicles that initiate endocytosis-most current evidence points to the cytoplasmic tails of endocytosed receptor proteins as possessing the information required for interaction with the coated pit region of the plasma membrane. In other words, a transport signal to leave the plasma membrane, rather than a retention signal to stay in the plasma membrane, appears to underlie selective endocytosis of surface proteins. More Than One Choice: The Simple Model Breaks Down The bulk flow/retention model described at the beginning of this review is applicable to a system of organelles strung together in a linear chain without branchpoints. If, however, at any point in the secretory pathway this system branches, then a choice must be made to stay in the or-

Minireview 705

ganelle or to go along one or another branches of the pathway. Such a situation could not be explained only by retention signals. It is difficult to determine whether any branchpoints exist within the central vacuolar system. One attractive place to postulate them is in the rrans-Golgillate endosomal network (Griffiths and Simons, 1986). It is in this region that newly synthesized proteins that reach the transQolgi are sorted to a number of possible endpoints including lysosomes, secretory granules, and plasma membrane-directed secretory vesicles. In addition, this may be the site within polarized epithelial cells at which proteins destined to be transported to different domains of the plasma membrane are sorted from each other. The acidic endosome is another likely candidate for a branchpoint within this vacuolar system (Geuze et al., 1983). Much of the fluid phase of the endosome along with some internalized membrane proteins is moved to lysosomes while other endocytosed membrane proteins are selectively returned to the plasma membrane. In addition, recent evidence points to the existence of resident proteins of the endosome not found (or found at much lower abundance) in the plasma membrane or the lysosome (Schmid et al., 1988). Thus components of the endosome can have at least three fates: retention, transport to lysosomes, or return to the plasma membrane. One possible site within the secretory pathway that may contain a branchpoint is the early part of the Golgi itself. As the initiating organelle in the movement of membrane through the secretory pathway, the ER is constantly losing lipid via transport to the Golgi; this lipid must be replaced. If, as has been suggested, the rate of loss of lipid from the ER vastly exceeds the rate of lipid biosynthesis, then the lipid must be retrieved (Wieland et al,. 1987). The question is whether a vesicular recycling pathway from the Golgi to the ER exists. The rapid movement of Golgi membrane proteins back to the ER observed in the presence of the fungal antibiotic brefeldin A has suggested the existence of just such a pathway (Lippincott-Schwartz et al., 1989) which we believe functions to return lipid to the ER. Our data suggest that resident membrane proteins of the Golgi are normally excluded from this recycling pathway, and we do not yet know which membrane protein components normally travel this retrograde path. Using brefeldin A we have begun to characterize the retrograde movement of proteins that are induced to enter this recycling pathway and can contrast this with the anterograde vesicular transport of proteins from the ER to the Golgi. Does the presence of a retrograde vesicular-transport loop from the Golgi to the ER threaten our picture of the efficient forward movement of fluid-phase markers (i.e., secreted proteins) through the secretory system? Lessons from the endocytic system, once again, suggest that this need not be the case. In the latter system there’is quite efficient sorting between recycling membrane components (receptors) and the fluid phase (as marked by the released ligands). One way to accomplish this is by segregating structures that maximize volume to surface area ratio from structures that minimize volume to surface area ratio, into which recycling receptors are concen-

trated. Accordingly, geometric considerations, at least in part, would allow for the transfer of volume to lysosomes and the recycling of certain membrane components to the plasma membrane. Perhaps similar geometric phenomena coupled with selective movement of membrane components into transport structures could allow efficient sorting of fluid-phase markers from membrane components at a number of places in the central vacuolar system. Mechanisms for Trafficking and Sorting Underlying the correct sorting of proteins within this complex vacuolar system is information contained within the sequence/structure of each protein that will determine its fate. This information can take two forms. First, as noted above, the protein may have a signal that is recognized within the cell by another protein which thereby determines the trafficking of the first protein. The effect of such recognition could be either retention or movement into a transport pathway. For the experimentalists, discerning these recognition signals may be relatively straightforward if the signals are discrete and well defined, such as mannose 6-phosphate; but, as in the case of translocation signal sequences or recognition sequences for coated pit-mediated endocytosis, the signal may be more elusive and loosely determined. The “signal” for interaction may be a general structural feature, such as an exposed hydrophobic face of a monomeric subunit of an oligomeric protein. The other form of information for intracellular trafficking does not involve the specific recognition by a receptor-like protein, but rather can be described as the possession of transport competency. This is analogous to the role of protein folding in determining the competency of a protein for translocation across a membrane (Meyer, 1988). Without knowing the structural requirements or constraints for entering transport vesicles within the secretory pathway, it is difficult to predict what form this transport competency would take. For example, the formation of higher-order aggregates may be either required or inhibitory for the entrance of proteins into transport vesicles such as those that allow transport from the ER to the Golgi. ‘This may be the mechanism underlying the correlation between the pathway taken by certain receptors in the endosome and the aggregation of these receptors (Mellman and Plutner, 1984). Failure to attain transport competence would then result in retention, not via the possession of specific retention signals, but by default. Retention alone cannot explain sorting at any branchpoints in the pathway; transport information, either in the form of specific signals or of transport competency, would be required to negotiate the sorting of membrane proteins through a branched pathway. At any branchpoint two different types of transport vesicles would have to arise from the parent organelle, and these vesicles would be targeted to distinct organelles. The targeting of sorted proteins could then be explained by different proteins satisfying the distinct molecular requirements for entering specific transport vesicles. That there can be an argument over whether trafficking and sorting within the secretory pathway rely on either retention signals or transport signals may reflect our de-

Cell 706

sire for simplifying generalizations more than the reality within the cell. It seems more likely that a multiplicity of sorting and trafficking mechanisms are utilized. Thus transport “incompetence” may be a mechanism that the cell uses to produce a resident membrane protein. Once a resident integral membrane protein exists for a particular organelle, however, interactive retention of other proteins becomes possible. The proposal of retention signals alone to explain the localization of all resident proteins in any organelle raises a classic “chicken and egg” problem: If all resident proteins of an organelle are localized by retention, how does such an organelle initially form? This may be more than a hypothetical question if organelles can be lost and reformed during the lifetime of a cell (Warren, 1985). In this case, how proteins are initially or primarily retained probably is closely connected to how the morphology and/or location of the organelle is determined. Thus one can imagine primary retention molecules as part of the underlying, nonorganellar organizing cytoskeleton that would establish, for example, the structure and location of a Golgi stack. Our problem, as we attempt to negotiate the conceptual maze of organelle traffic, is how do we recognize the signals that control the traffic, and once we recognize them, how do we determine whether those signals func-

tion by telling a protein either “you can’t go” (transport incompetence), “you must stay” (retention), or “you must go” (transport signal)? References Geuze, H. J., Slot, J. W., Strous, G. J. A. M., Lodish, Schwartz, A. L. (1983). Cell 32, 277-287.

H. F., and

Goldstein, J. L., Brown, M. S., Anderson, R. G. W., Russell, D. W., and Schneider, W. J. (1965). Annu. Rev. Cell Biol. 7, l-40. Griffiths,

G., and Simons, K. (1986). Science 234, 438-443.

Lippincott-Schwartz, J., Yuan, L. C., Bonifacmo, R. D. (1989). Cell 56, 801-613. Mellman,

J. S., and Klausner,

I., and Plutner, H. (1984). J. Ceil Biol. 98, 1170-1177.

Meyer, D. I. (1988). Trends Biochem.

Sci. 73, 471-474.

Munro, S., and Pelham. H. R. B. (1987). Cell 48, 899-907. Paabo. S., Bhat. B. M., Wold, W. S. M., and Peterson, P A. (1987). Cell 50, 311-317. Palade, G. E. (1975). Science

789, 347-358.

Pfeffer, S. R., and Rothman, 829-852

J. E. (1987). Annu. Rev. Biochem.

56,

Rose, J. K., and Doms, R. W. (1988). Annu. Rev. Cell Biol. 4, 258-289. Schmid, 73-83.

S. L., Fuchs, R., Male, F’., and Mellman,

Warren, G. (1985). Trends Biochem. Wieland, F. T., Gleason, (1987). Cell 50, 289-300.

I. (1988). Cell 52,

Sci. 70, 439-443.

M. L., Serafini,

T. A., and Rothman,

J. E.