Talking point Amphitropic proteins: a new class of membrane proteins

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Features Talking Point Amphitropic proteins: a new class of membrane proteins Paul Bum Spedfic lipidsplay crucial roles in signal transmission across membranes and in the modulation, regulation and membrane.association of proteins. Severalproteins, previously described as soluble cytoplasmicproteins, appear to interactboth specifically with h'pidsand directly with the hydrophobic part of membranes, indicating a reversible association of theseproteins with membranes under appropriate conditions. Here it is proposed that such proteins belong to a new class of membrane proteins defined as the 'amphitropic' proteins. From this new concept emerge important implications for studies of cytoskeleton-membra~ interactions, transmembrahe signaling, and the orgardzation of the cytoskeletotl. Only very recently have lipids been recognized to play important roles in the association of proteins with membranes TM. Noncovalent and covalent interactions between lipids and proteins have been reported. Specific lipids, such as long-chain fatty acids and phosphatidylinositol-derived lipids, have been found to interact with a number of proteins such as the oncogene product pp60 src, a fatty acid-acylated, tyrosine kinase; protein kinase C, a diacy!glycerol-activated, Ca 2+- and phospholipid-dependent serine/threonine kinase; and many cytoskeletal proteins (see Table I). In all these cases binding of lipids may be one of the mechanisms involved in the reversible association of these proteins with membranes. In addition, in the case of cytoskeletal proteins, lipid-protein interactions may represent a driving force for the assembly, organization and rearrangement of cytoskeletal elements (see below). In the first part of this article actinassociated cytoskeletal proteins that ineract with specific lipids and/or membranes are briefly introduced. A new class of membrane proteins which includes, but is not restricted to, cytoskeletal proteins is then defined. Implications emerging from this new concept with particular regard to the dynamic association of cytoskeletal elements with membranes, and possible functional relationships between the phosP. Burn is at the Department of Biology, Uni:,ersityof California at San Diego, La Jolla, CA 92093, USA.

phatidylinositol cycle, amphitropic proteins and the regulation of cytoskeletal organization, are discussed in the last part of the article. Interaction of cytoskeletal proteins with specific lipids and membranes Noncovalent interactions. Several cytoskeletal proteins have been demonstrated to interact with specific lipids and/or membranes in a noncovalent manner, both in vitro and in situ. Examples, summarized in Table I, include: (1) the cytoskeletal linker proteins ¢t-actinin and vinculin, both of which were suggested to be involved in anchoring of actin filament bundles to membranes; (2) the actinsequestering protein profilin; (3) the actin filament end-blocking protein gelsolin; (4) the Ca 2+-, phospholipid- and actin-binding protein p36 (also referred to as p34, p39, protein I or calpactin); (5) the actin-crossfinking protein spectrin; (6) the erythrocyte protein 4.1; and (7) a 110 kDa microvillar protein. Covalent interactions. Recently, the covalent modification of several cytoskeletal proteins with lipids have been reported. Among them are vinculin, p36 actin, ankyrin and protein 4.1 (Table I). They were all found to be modified by long-chain fatty acids such as myristic acid or palmitic acid. Future studies may identify other cytoskeletal proteins which contain covalently bound lipids. The cytoskeletal linker proteins ~t-actinin and talin seem to be potential candidates for this type of covalent modification, since both of which were suggested

to be involved in anchoring of actin filament bundles to membranes. The above cytoskeletal proteins (Table I), which were all previously considered to be cytoplasmic proteins, appear to interact either covalently or noncovalently with specific lipids and/or directly with the hydrophobic core of membranes, in vitro and/or in vivo. In most cases not only was a preference for specific lipids found, but lipid-binding domains on the proteins were identified or a functional regulation of the proteins by lipids was demonstrated (e.g. ~t-actinin, profilactin, gelsolin), indicating the relevance of these specific interactions. Whether the association of these proteins with specific lipids also implies an association with membranes, and the nature of the interaction of these proteins with membranes have yet to be determined in every individual case. In a few cases (e.g. a-actinin, vinculin) the direct insertion of these protein~ into the hydrophobic domain of membranes has been reported. It is also important to determine whether these interactions of proteins with specific lipids and membranes occur in vivo. To date, all the experiments which have demonstrated a covalent modification of cytoskeletal proteins witl-: lipids were performed in living cells. In addition, convincing evidence exists that noncovalent interactions between aactinin and specific lipids occur in living blood platelets. However, further experiments in situ and in vivo are needed to confirm the other examples where interactions of proteins with lipids or membranes in vitro have been reported. A new class of membrane proteins Proteins involved in connections between the intracellular and extracellular spaces are usually described as integral membrane components 5. Alternatively, peripheral membrane proteins are proposed to mediate interactions between membrane components and either the cytoplasmic or extraceUular compartment. It is believed that peripheral proteins located on the cytoplasmic side of the membrane bind to the exposed hydrophilic domains of particular integral proteins without having any ~) 1988. Elsevier Pubfications Cambridge 0376- 5067/88/$02.00

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modification of those pn)teins with lipids might be important. "D~e addition of a Cytoskeletal protein Lipida Refs hydrophobic lipid tail to a cytoplasmic, Noncovalent interactions water-soluble protein has been shown to u-Actinin DG, FA b-g result in its membrane association 3. Profilin/Profilactin PIP 2, PIP h, i Removal of the lipid from the protein Gelsolin PIP2 j could reverse this effect. Such a Vinculin Acidic PL (e.g. PI, PIP 2, PS) k, I mechanism could be involved in the p36 (subunit of Protein I, Calpactin) PS, PI m, n dynamic and reversible process of Spectrin PS, PE o Protein4.1 PS p anchoring cytoskeletal elements to Protein 4.1-Glycophorin PIP2, PIP q membranes in living cells. Alternatively, 110 kDa Microvillar protein PL r, s amphitropic proteins may contain a Covalent interactions covalently bound lipid that is buried Vincufin Pal, Myr t, u inside the hydrophobic part of the folded p36 (subunit of Protein I, Calpactin) Myr v protein. A reversible conformational Actin (Dictyostelium discoideum) Pal w change induced, for example, by phosAnkyrin Pal x,y phorylation, could lead to the exposure Protein 4.1 Pal, Ste, Ole z of this hydrophobic region on the protein a-Actinin ? surface and provide the driving force for Tafin ? membrane insertion. However, quesaAbbreviations: DG, diacylglycerol; FA, fatty acids; PL, phospholipids; PI, phosphatidylinositol; PIP, tions remain such as: (1) Does this type phosphatidylinositol 4-phosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; PS, phosphatidylserine; of amphitropic protein bind to the memPE, phosphatidylethanolamine. Myr. myristic acid; Pal, palmitic acid; Ste, stearic acid; Ole, oleic, acid. brane only via hydrophobic lipid tails or References: b Burn, P., Rotman, A., Meyer, R. K. and Burger, M. M. (1985) Nature 314, ,59-472. c Burn, P. (1987) in Membrane Proteins: Proceedings of the Membrane Protein Symposium (Goheen, do parts of the proteins insert into the S. C., ed.), pp. 747-763, Bio-Rad Laboratories. d Burn, P. in UCLA Symposia on Molecular and Cellular hydrophobic domain of the membrane Biology (Satir, P., Condeelis, J. and Lazarides, E., eds), Alan R. Liss, Inc. (in press), e Meyer, R. K., as well? (2) Are there specific proteins in Schindler, H. and Burger, M. M. (1982) Proc. Nail Acad. Sci. USA 79, 4280--4284. f Rotman, A., membranes that have a selective affinity Heldman, J. and Linder, S. (1982) Biochemistry 21,1713-1720. g Burn, P. (1988) J. Cellular Biochem. 36, 15-24. h Lassing, I. and Lindberg, U. (1985) Nature 314, 472-474. i Lassing, I. and Lindberg, U. J. Cellular for acylated proteins? (3) What enzymes are involved in possible modification Biochem. (in press), j Janmey, P. A. and Stossel, T. P. (1987) Nature 325, 362-364. k Ito, S., Werth, D. K., Richert, N. D. and Pastan, I. (1983) J. Biol. Chem. 258, 14626-14631. I Niggli, V., Dimitrov, steps? Both lipid-protein and proteinD. P., Brunner, J. and Burger, M. M. (1986)./. Biol. Chem. 261,6912--6918. m Geisow, M. J. and Walker, protein interactions may be important in J. H. (1986) Trends Biochem. Sci. 11,420-423. n Glenney, J. (1986) J. Biol. Chem, 261, 7247-7252. the association of amphitropic proteins o Cohen, A. M., Liu, S., Derick, L. H. and Palek, J. (1986) Blood 68,920-926. p Sato, S. B. and Ohnishi, with membranes. S. (1983) Fur../. Biochem. 130, 19-25. q Anderson, R. A. and Marchesi, V. T. (1985) Nature 318, 295It does not necessarily need to be a 298. r Glenney, J. R. and Glenney, P. (1984) Cell 37,743-751. s Conzelman, K. A. and Mooseker, M. S. modification of the amphitropic protein (1986) J. Cellular Biochem. 30, 271-279. t Burn, P. and Burger, M. M. (1987) Science 235, 476-479. u itself that triggers a membrane associaKellie, S. and Wigglesworth, N. M. (1987) FEBS Left. 213,428-432. v Soric, J. and Gordon, J. A, (1985) tion. The induction of local changes in Science 230, 563-566. w Stadler, J., Gerisch, G., Bauer, (3. and Deppert, W. (1985) EMBO l., 4, 11531156. x Staufenbiei, M. (1987) Mol. Cell. Biol. 7, 2981-2984. y Staufenbiel, M. and Lazarides, E, (1986) the lipid composition within the memProc. NatlAcad. Sci. USA 83,318-322. z Keenan, T. W. and Heid, H. W. (1982)Eur../. Cell. Biol. 26, brane could effect an association as well. 270-276. For instance, local changes in the lipid direct contact with the hydrophobic part the amphitropic proteins. This concept composition, restricted to membrane of the membrane. The association of suggests the possibility of translocation areas where a signal is induced, could (cytoplasmic) actin filaments with mem- of amphitropic proteins from the cyto- lead to a directed insertion of an amphibranes seems to involve integral and plasmic compartment into the membrane tropic protein to that site. This non-ranperipheral membrane proteins and was compartment of cells and vice versa. It dom, directed insertion would have the therefore considered as a typical exam- further suggests a transfer of information additional advantage that no lateral ple of this type of interaction (see Fig. via specific lipids. These lipids may pro- movement of the protein within the 1A and below). vide a signal for lipid-protein interac- plane of the membrane would be needed However, many proteins, including tions to occur and may thereby represent to bring the protein to its place of funcprotein kinase C, pp60src and all the a driving force for the dynamic associa- tion. cytoskeletal proteins mentioned above tion of amphitropic proteins with memThe cytoskeletai linker proteins vin(Table I), cannot be simply classified as branes (see below). culin 20 and u-actinin 21-24 are two examcytoplasmic, peripheral or integral memples of amphitropic proteins that appear to brane proteins. These proteins can be Possible mechanisms for the reversible interact reversibly with the hydrophobic isolated either in a soluble cytoplasmic insertion of amphitropic proteins into the part of membranes. The insertion of vinform or associated with membranes, pre- hydrophobic domain of membranes culin into the hydrophobic domain of the sumably as a result of specifically bound A crucial question concerning the membrane may involve a mechanism lipid. It appears that they can exist in a interaction of amphitropic proteins with similar to the first type described above, cytoplasmic soluble form as well as the hydrophobic domains of membranes whereas the insertion of u-actinin into in a membrane embedded form, thus is what might trigger the translocation of membranes may involve a mechanism exhibiting properties of cytoplasmic, these proteins from the cytoplasmic similar to the latter type. Although it peripheral and integral membrane pro- compartment into the membrane com- might be too early to generalize these teins. It is proposed, therefore, that such partment? In the case of proteins concepts, future studies concentrating proteins belong to a newly recognized containing covalently bound lipid, a on possible regulatory mechanisms class of membrane proteins defined as mechanism involving post-translational involved in the formation of cytoskeleTable L Cytoskdetal proteins that have been demonstrated to interaa with specific lipids

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ton-membrane associations can "be encouraged by the fin&rigs described above.

Cytoskeleton-membrane associations: specific linker proteins and different modes of interaction with membranes The cytoskeleton, con:;isting of actin filaments, microtubles and intermediate filaments, is the most prominent component of the cytomatrix of eukaryotic cells. Cell properties such as shape, internal organization and movement depend upon these complex networks of protein filaments. Common biological phenomena, including cell division, cellcell interactions, cell adhesion and migration all involve cytoskeletal elements. They participate in many physiologically important processes such as embryological development, wound healing, immune and non-immune defense mechanisms and oncogenic transformation. A rapid reorganization of cytoskeletal elements has been demonstrated in response to certain growth factors and tumor promoters and during cell transformation6, 7. Growth-related cellular functions appear to be regulated by signals which are transmitted through an organized cytoskeleton. It is thought that the cytoskeleton is also involved in the transfer of information from the plasma membrane to the nucleus since important cellular processes such as gene expression and cellular differentiation are affected by the organization of the cytoskeletonS. 9. Nevertheless, some of the most crucial questions concerning cytoskeletal organization remain unanswered. How do cytoskeletonassociated proteins, and therefore the cytoskeleton itself, interact with membranes? How are associations between cytoskeletal proteins and membranes regulated? What are the mechanisms involved in the dynamic association of cytoskeletal elements with membranes? The most widely accepted view of the association of cytoskeletal elements with membranes is that specific linker proteins (cytoplasmic/peripheral membrane components) mediate either a direct or indirect interaction of actin filaments (cytoplasmic components) with transmembrane receptors (integral membrane components) (Fig. 1A). A single linker protein or a chain of several different linker proteins may be involved in these associations (Fig. 2A). This view implies that the linker proteins interact with the cytoplasmic domain of a transmembrane receptor without having any direct contact with the hydrophobic part of the membrane (Figs 1A, 2A). How-

ever, the studies with cytoskeietal proteins discussed above, suggest that at least some linker proteins (amphitropic proteins), may interact directly s~dtl~the hydrophobic domain of membranes (Figs 1B, 2B). This view by no means excludes an additional requirement of integral transmembrane proteins in cytoskeleton-membrane associations. They remain necessary for a continuous transmembrane connection between the intracellular and extracellular spaces (Figs 1C, 2C), and may even be necessary for establishing or stabilizing interactions between iipids ~ad cytoskeletal linker proteins. Several different types of cytoskeletonmembrane associations have been described in various cell types which may involve distinct linker proteins. Studies with lymphocytes, where the binding to susceptible target cells1° and the capping of surface proteins by antibodies 11 resulted in a segregation of different linker proteins suggest that different mechanisms of actin filament anchoring at membranes may occur even within the same cell. Thus, in different cell types or even within the same cell, actin filamentmembrane linkages might be mediated by different linker proteins (Fig. 2). In addition, different modes of interaction of linker proteins with membranes seems probable. For example, a direct interaction of the linker proteins with the hydrophobic part of the membrane (Figs 213, 2C) or an indirect interaction via the cytoplasmic domain of transmembrane receptors (Fig. 2A) may occur. "Hiecomplex and heterogeneous nature of cytoskeleton-membrane associations suggests that both modes of interaction may occur in living cells. Moreover, it is tantalizing to envisage both modes of association co-existing within the same cell.

may include tyrosine phosphorylation and/or serine/threonine phosphorylation of proteins as suggested by studies of integlin-talin interactions in living lymphocytes 11. All of these different mechanisms involve enzymatic activities that act either on the lipid bilayer or directly or indirectly on the proteins that participate in these associations. These metabolically regulated, biochemical mechanisms could therefore provide a means for the control of dynamic cytoskeleton-membrane interactions/n vivo. This new concept of metabolically regulated cytoskeleton-membrane associations has important consequences in cell physiology. An appropriate signal could induce a unique mechanism that acts on a single linker protein involved in a particular type of cytoskeleton-membrane association. Distinct signals may utilize different mechanisms that act on specific linker proteins. Different associations between cytoskeletal elements and membranes that co-exist within the same cell may therefore be regulated and modulated independently.

Functional relationship between the phosphatidylinositol cyde, amphitropic proteins and cytoskeletal organization Ligand-receptor interactions, which in many different cell systems lead to an increased activity in the phosphatidylinositol cycle, are accompanied by an increased formation of actin filaments. The activation of platelets with thrombin, for example, was shown to be linked to an increased turnover of phosphatidylinositol (PI) leading to the formation of two second messengers: diacylgiycerol (DG) and inositol 1,4,5trisphosphate (IP3) (for reviews see Refs 1, 2, 15 and 16). DG operates within the plane of the membrane where it is thought to be important in the activation of the Ca2+- and phospholipid-dependent Metabolically regulated associations of protein kinase C. IP3 is released into the the eytoskdeton with membranes Although the interactions between the cytoplasm and functions in the mobilizacytoskeleton and membranes7,m14.2s tion of intracellular Ca 2+. In addition, have been studied for many years, little is direct analysis of the percentage of known about how these associations are G- and F-actin in platelet extracts regulated. However, the existence of demonstrated that, following stimulaamphitropic linker proteins and their tion, a rapid polymerization of actin and reported interaction with specific lipids a reorganization of actin filaments into and membranes indicates that cytoskele- bundles occurs t7` ts. Several reports proton-membrane associations may be vide evidence for a specific effect of PImetabolically regulated. Covalent mod- derived lipids and DG on tile actin-bmdifications of these proteins with lipids or ing proteins profilactin, gelsolin and ctenzyme-induced local changes in the actinin (Table I). Thus, it is tempting to lipid composition of membranes could suggest that the formation of actin filaeffect the reversible association of cyto- ments as well as their association with skeletal proteins with membranes. Ot~her membranes may be regulated through enzyme-catalysed mechanisms involved the phosphatidylinositol cycle19.23. On the basis of these findings, a posin the regulation of these associations

82

l~g. 1. Three alternativepossibilitiesfor the interaction of linker proteins (L ) with membranes. The anchoring of actin filaments (A) to the membrane is shown via a chain of three intervening linker proteins (L !, L z, Lj). (A) The linker protein (L I), is shown as a cytoplasmic/peripheral membrane protein that interacts with the exposed hydrophilic domain of a particular integral membrane protein (R). Note, none of the linker proteins is in direct contact with the hydrophobic part of the membrane. (B) The linker protein (L*) is shown as an amphitropic membrane protein that is in a direct contact with the hydrophobic part of the membrane. (C) The linker protein (LI") is shown as an amphitropic membrane protein that is in a direct contact with both the hydrophobic part of the membrane and an inte~al membrane protein (R).

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proteins into the hydrophobic domain of membranes (see above). The cytoskeletal protein a-actinin is shown as an amphitropic linker protein that can exist in a cytoplasmic (L) as well as in a membrane embedded (L*) form (Fig. 3). Questions that remain to be resolved include whether integral membrane proteins are involved in mediating a first contact between a-actinin and the lipids in the membrane, or whether they are involved only in later stages, such as the stabilization of the newly formed cytoskeletonmembrane associations. However, a mechanism for the formation and membrane association of actin filaments as described in Fig. 3 seems to be likely at least in blood platelets. Whether a similar mechanism for the regulation of cytoskeletal organization exists in other cell types as well, still remains to be answered.

Conclusions A growing number of reports show that many proteins, previously thought to be soluble cytoplasmic proteins, interact with specific lipids/n vitro as well as/n s/tu. Under appropriate conditions sible mechanism for the formation of actin rides a functional relationship between direct interactions of several of these filaments and their dynamic association transmembrane signals, the phosphatidyl- proteins with membranes occur. The with membranes may involve the phos- inositol cycle, amphitropic proteins and nature of these interactions, in particular phatidylinositol cycle and the amphi- the organization of the cytoskeleton. It is whether these proteins insert directly tropic proteins ~t-actinin, gelsolin and also an example of how enzyme-catalysed into the hydrophobic part of the bilayer, profilactin. A possible sequence of re- local changes in the lipid composition is not yet clear in all cases and should be actions is illustrated in Fig. 3. This within membranes could be involved in examined in future experiments. Howmetabolically regulated mechanism pro- the reversible insertion of amphitropic ever, examples of cytoskeletal proteins such as a-actinin and vinculin clearly demonstrate that direct contacts between the hydrophobic domain of membranes and proteins occur. These proteins are therefore proposed to be members of a new class of membrane proteins, the amphitropic proteins, which, under appropriate conditions, can exist in either a soluble cytoplasmic or a membrane embedded form. This new class of membrane proteins is not restricted to cytoskeletal proteins. This reappraisal adds a new dimension to the field of cytoskeleton-membrane associations. In addition to the wellaccepted membrane anchoring of the cytoskeleton through the cytoplasmic domain of integral membrane receptors, a second type of linkage involving a direct insertion of cytoskeletal proteins into the hydrophobic part of the memFig. 2. Aiternative possibilities for actin filament-membrane associations involving different linker proteins brane may exist. Thus not only protein(L ) and different modes of interaction. The anchor~'zg of actin filaments (A ) to the membrane is shown either protein interactions, but also lipidinvolving a single linker protein (LI) or a choir: to two intervening linker proteins (Lz, Lz). (A) The two protein interactions may be crucial in linker proteins (L 1) and (L z) are shown as cytoplasmiclperipheral membrane proteins that interact with the the association of cytoskeletal proteins exposed hydrophilic domain of two different integral membrane proteins (R 1) and (R 2). Neither of the linker with membranes. proteins is in a direct contact with the hydrophobic part of the membrane. (B) The two linker proteins (LI") Cells appear to be provided with sevand (Lz') are shown as amphitropic membrane proteins that are in a direct contact with the hydrophobic part eral mechanisms with which to regulate of the membrane. (C) The two linker proteins (LI*) and (L2*) are shown as amphitropic membrane proteins the association of cytoskeletal proteins that are in a direct contact with both, the hydrophobic part of the membrane, and two different integral memwith membranes. These may include (1) brane proteins (RI) and (R2). Note, all types of associations illustrated may co-exist within the same cell.

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membrane associations, transmembrane signafing and the regulation of cytoskeletal organization. Thus, future research may lead to a better understanding of dynamic and complex processes such as cell motility and cell transformation. Acknowledgements I am most grateful to my colleagues R. Shimonkevitz, N, Colley, D. Wegmann and G. Cox for critical and constructive comments on this manuscript. I thank Professor D r Max M. Burger for many stimulating discussions on the subject of this article and Professor D r S. J. Singer for giving me the opportunity of working in his laboratory. My EMBO postdoctoral fellowship (ALTF:308--1985) is acknowledged. Fig. 3. A possible mechanism for theformation and dynamic association of actinfilaments with membranes involving amphitropic proteins and the phosphatidylinositol cycle. Receptor (Rl)-mediated stimulation (S) of cells leads to the well-documented increase in the turnover of phosphatidylinositol (PI). Phosphorylation of (PI) to phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIPz) initiates the dissociation of profilactin and gelsolin--actin complexes and promotes the nucleated assembly of actin by the specific interaction of PIPz with these proteins. The receptor-mediated stimulation of cells also activates phospholipases (PL C and PL A z) by mechanisms probably involving G proteins (G). The phospholipases initiate the cleavage of PIP2 or other phospholipids (PL) which leads to the generation of dlacylglycerol (DG), fatty acids (FA) and inositol 1,4,5-trisphosphate (IP~. Protein kinase C (PKC) is activated and Ca2+ is releasedfrom internal stores. Formation of DG and FA causes local changes in the lipid composition of the membrane, which tn'ggers the translocation of cytoplasmic oL-actinin (L) into the hydrophobic domain of the membrane. Complex formation between a-actinin, DG, and FA may lead not only to a direct insertion of a-actinin into the hydrophobic part of the membrane, but may also increase the affinity and attachment of membrane-embedded mactinin (L*) with the actin filaments (,4). In addition, direct interactions between a-actinin--lipid complexes (L*) and integral membrane proteins (Rz) may be established.

the covalent modification of cytoskeletal proteins with lipid, (2) the induction of local changes in the lipid composition within membranes and (3) tyrosine as well as serine/threonine phosphorylation of proteins. It appears that cytoskeleton:,~embrane associations could be raetabolicaUy regulated. The phos-

phatidylinositobderived fipids may play a particularly crucial role in the formation and dynamic membrane association of actin filaments and may thereby be involved in the regulation of cytoskeletal organization.

These viewpoints suggest new directions for the study of cytoskeleton-

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References

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