A new study from the lab of Bart De Strooper and colleagues at the University of Leuven, Belgium, adds γ-secretase to the list of proteins that interact with tetraspanins, a large family of small, ubiquitous proteins that spin a web of lateral associations between transmembrane proteins. Named because they cross the cell membrane four times, tetraspanins regulate a myriad of cell processes that involve cell adhesion, signaling, and proteolysis (for a recent review, see Charrin et al., 2009). In a paper published online on October 18 in Nature Cell Biology, De Strooper and colleagues report finding multiple tetraspanins in a proteomic screen for γ-secretase-interacting proteins. They show that tetraspanins help regulate γ-secretase activity, and demonstrate that disruption of the tetraspanin web inhibits amyloid-β (Aβ) production in cultured cells.

“Overall, these findings are important contributions to our understanding of the cell biology of γ-secretase, identifying partner proteins that can modulate protease activity and affect amyloid production,” wrote Michael Wolfe, Harvard Medical School, in an e-mail to ARF. “Going forward, it will be worthwhile to investigate possible roles of tetraspanin genes in the pathogenesis of AD or whether the levels of the encoded proteins are otherwise altered,” said Wolfe, who was not involved in the work (see complete text of Wolfe comment below).

To be active, the γ-secretase complex must contain four proteins, the presenilins (PS1 or PS2), nicastrin, Aph1, and Pen-2. In the new study, first author Tomoko Wakabayashi went fishing for other members in a γ-secretase preparation from cultured cells. The researchers expressed doubly affinity-tagged PS1 or PS2 in presenilin knockout mouse embryo fibroblasts, and then purified the complex over tandem affinity columns. Mass spectrometry analysis of the resulting mixture yielded 59 proteins that co-purified with either PS1 or PS2, but not with the PS homolog SPPL3. All 46 of the proteins that reproducibly co-purified with PS2 were also found with PS1, suggesting that the two presenilins associate with a similar range of proteins. Among the 46 were some familiar proteins including Tmp21 (see ARF related news story), as well as some new players. The novel finds included the tetraspanins CD81 and Upk1b, and a tetraspanin partner protein, the cell surface immunoglobulin superfamily protein EWI-2. Coimmunoprecipitation experiments confirmed the association between γ-secretase and CD81, and identified additional interactions with a third tetraspanin, CD9, and a second EWI protein, EWI-F.

The tetraspanins play a role in γ-secretase function, as indicated by RNAi knockdown and overexpression experiments. Depletion of CD81, EWI-F, or the tetraspanin-associated protein CD98hc decreased production of Aβ in HEK293 or Hela cells. Cells from CD81 or CD9 knockout mice showed an increase in C-terminal fragments of endogenous γ-secretase substrates including the amyloid precursor protein, suggesting that in the absence of the tetraspanins, either the activity of γ-secretase or its access to substrates is disrupted. Consistent with this idea, HEK293 cells treated with anti-CD9 monoclonal antibodies had their Aβ production reduced by more than half.

Cell fractionation experiments supported the idea that γ-secretase localizes to tetraspanin-enriched membrane domains (TEMs). The tetraspanins and γ-secretase proteins co-migrate in the low-density fractions of sucrose density gradients, the researchers show. Dissociation of TEMs by detergent treatment also resulted in loss of presenilin from the same fraction. Previously, γ-secretase as well as β-secretase have been localized to lipid rafts (Wahrle et al., 2002, and see ARF related news story on Hattori et al., 2006), which are detergent-insoluble, cholesterol-rich membrane microdomains similar to TEMs. However, TEMs are different from lipid rafts in their protein and lipid makeup and in their detergent sensitivity. (There are no tetraspanins in lipid rafts, for instance.) The authors propose that the association with TEMs might account for a large part of the γ-secretase activity previously attributed to lipid rafts.

The work raises many questions: Does the disruption of the tetraspanin interactions affect γ-secretase localization, stability, activity, or all of the above? Are the interactions and functional effects direct or indirect? Do tetraspanins contribute to AD pathology, and might they make plausible drug targets? Tetraspanins have been previously shown to regulate the activity of an α-secretase, ADAM10 (Xu et al., 2009), suggesting that these little proteins may hold more than one clue to Aβ production in their tangled web.—Pat McCaffrey


  1. Another beautiful work on the biochemistry of γ-secretase complex from the group of Bart de Strooper.

    This connection to tetraspanins offers key insights into the "raft"
    association of γ-secretase. The raft link to APP processing is very weak and highly contended. Lipid rafts are dynamic platforms that could be, to a large extent, biochemically purified by detergent extraction methods.

    Typically Triton X-100 (in concentration ranging from 0.3 percent to 1 percent) insoluble fractions of membranes represent the raft fraction. However, tetraspanin microdomains (TEM) differ from the "canonical" rafts in that these are triton soluble but are insoluble to other milder detergents such as CHAPS or BRIJ. This study clearly demonstrates a direct link between tetraspanin microdomains and γ-secretase, thereby providing evidence that much of the γ-secretase activity is probably confined to tetraspanin microdomains.

    An (obvious) interesting question is, How about Notch cleavage? While γ-secretase cleavage of β-CTF seems to depend on its interaction with the tetraspanin web, it would be interesting to see if there is a lateral compartmentalization in γ-secretase (non-TEM vs. TEM γ-complexes) that could determine substrate specificity. In my opinion, this systematic interactome analysis elegantly shows that Aβ production, though primarily achieved by only three players (APP, BACE1, and γ-secretase complex), is far too complex in the cellular context.

    View all comments by Lawrence Rajendran
  2. This is an excellent study by the De Strooper lab, using a biochemical approach to identify proteins that associate with the γ-secretase complex. The work was rigorously done, with important controls to identify interactions specific to γ-secretase (e.g., parallel purification of the presenilin-like protease SPPL3). The authors confirm several proteins previously reported to interact with γ-secretase, such as TMP21, β-catenin and Rab11, further strengthening confidence in their method. Most interesting was the identification of tetraspanin proteins, which are important in many basic cellular activities, including formation of certain lipid raft-like microdomains. Indeed, γ-secretase components co-distribute with tetraspanin proteins in a sucrose density gradient.

    Knockdown of certain associated tetraspanin-web proteins by RNAi led to modest but significant decreases in amyloid-β-protein levels and increases in APP C-terminal fragments that are γ-secretase substrates. Conversely, overexpression can lead to increased amyloid production.

    Overall, these findings are important contributions to our understanding of the cell biology of γ-secretase, identifying partner proteins that can modulate protease activity and affect amyloid production. Going forward, it will be worthwhile to investigate possible roles of tetraspanin genes in the pathogenesis of AD or whether the levels of the encoded proteins are otherwise altered. Targeting tetraspanins to prevent or treat AD, however, is probably not advised, because these proteins affect other γ-secretase-mediated proteolytic events (syndecan-3, N-cadherin, APLP-2, and ADAM10, shown in this new report); they are probably also critical for Notch proteolysis and signaling. Inhibition of Notch signaling leads to toxic effects that must be avoided in targeting γ-secretase, whether directly or indirectly, for AD.

    View all comments by Michael Wolfe
  3. This represents a careful and significant study with clean results. It probably explains why γsecretase loses activity in Triton-X100 and helps in a more thorough functional characterization of the enzyme.

    View all comments by Kumar Sambamurti

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News Citations

  1. Eibsee: Still Game for γ—Sparring With a Formidable Enzyme
  2. Chew ’em Up and Spit ’em Out: Aβ Leaves Cells via Exosomes

Paper Citations

  1. . Lateral organization of membrane proteins: tetraspanins spin their web. Biochem J. 2009 Jun 1;420(2):133-54. PubMed.
  2. . Cholesterol-dependent gamma-secretase activity in buoyant cholesterol-rich membrane microdomains. Neurobiol Dis. 2002 Feb;9(1):11-23. PubMed.
  3. . BACE1 interacts with lipid raft proteins. J Neurosci Res. 2006 Sep;84(4):912-7. PubMed.
  4. . Tetraspanin12 regulates ADAM10-dependent cleavage of amyloid precursor protein. FASEB J. 2009 Nov;23(11):3674-81. PubMed.

Further Reading

Primary Papers

  1. . Analysis of the gamma-secretase interactome and validation of its association with tetraspanin-enriched microdomains. Nat Cell Biol. 2009 Nov;11(11):1340-6. PubMed.