We addressed the question whether MARCH family proteins were functionally related to viral immune evasion proteins of the K3 family. The overall sequence homology of these predicted proteins is rather low and limited mostly to the RING-CH domain (Fig. ). The primary structure was thus suggestive but insufficient for us to conclude which of these proteins were functionally related to the viral immunomoldulators. Using a panel of potential substrates selected as known substrates of viral K3 family proteins, we observed an efficient and specific downregulation of various cell surface glycoproteins by the cellular homologues. The partially overlapping, partially distinct substrate specificity observed for the MARCH proteins is also typical of the viral proteins. The molecular reason for this specificity is not known, but substrates seem to interact transiently with the viral proteins (20
) and substrate recognition seems to reside in the transmembrane regions of the targets (11
). Consistently, we observed that sequence correlation in the RING-CH and transmembrane domains correlated with substrate specificity, whereas deletion of the amino-terminal or carboxy-terminal domains did not affect substrate downregulation.
Our results also confirm the previously reported downregulation of B7.2 by MARCH-VIII (c-MIR) (16
). However, we observed that MARCH-I, which is homologous to MARCH-VIII, also efficiently downregulated B7.2 and that both MARCH-I and VIII also downregulated Fas and Tfr. Moreover, the unrelated MARCH-II also downregulated B7.2. In contrast to the findings of Goto et al., we did not observe a preferential expression of MARCH-VIII in lymph nodes. Rather, MARCH-VIII mRNA was found at low levels in most tissues examined (Fig. ). Also different was our observation of a moderate, but significant downregulation of transfected HLA-A2.1 by MARCH-VIII. This could be caused by a higher level of expression achieved in our study with the tetracycline-inducible system which might facilitate a weak interaction. In general, however, a pattern emerged whereby MARCH-VIII and MARCH-I targeted a distinct set of surface glycoproteins compared to MARCH-IV and -IX. This pattern mimics the dichotomy of function for the K3 and K5 genes of KSHV. Although K3 downregulates a broader spectrum of MHC-I alleles compared to K5 (24
), K5 but not K3 also downregulates B7.2 (10
The essential, sequence-independent role of lysines in the cytoplasmic tails of substrates together with ubiquitinated HLA-A2.1 strongly suggests that ubiquitination is required for downregulation by the MARCH family. In contrast to the study by Goto et al., who reported multiple ubiquitination of B7.2, we observed only a single species of ubiquitinated HLA-A2.1. This difference could be caused by the fact that B7.2 contains 11 lysines in its cytoplasmic tail (10
), whereas the HA-tagged A2.1 form used in our experiments contained only two lysines (31
). Recently, it was shown that multiply ubiquitinated receptor tyrosine kinases did not contain polyubiquitin but were monoubiquitinated at multiple sites (18
). Thus, it is possible that B7.2 contains monoubiquitins at multiple sites, whereas only one of the two lysines is used for ubiquitination of the HA-tagged version of HLA-A2.1. Notably, only two ubiquitinated forms of murine MHC-I molecule Db, which contains three lysines, were found in MHV68-K3 transfected cells (6
). A central role for ubiquitin is further corroborated by the observed ubiquitin ligase activity of the RING-CH domain in vitro and by the partial restoration of MHC surface expression by GFP-Vps4-E228Q. Very similar observations were reported for both herpesviral and poxviral K3 family proteins, which were shown to require at least one lysine in the tail (6
), and substrate internalization was inhibited by interference with the MVB pathway (20
). For both KSHV-K5 and MV-M153R, it was also shown that the isolated RING-CH domain acts as ubiquitin ligase (11
). The current model for the function of the viral proteins is that they transiently interact with their substrates and mediate the ubiquitination of the cytosolic tail. Our data, as well as those of Goto et al. (16
), are consistent with the cellular homologues acting in a manner very similar to that of their viral relatives.
Although MARCH proteins induced the internalization of glycoproteins at the cell surface, subcellular localization studies revealed that these proteins all localize to intracellular membrane compartments. Similarly, all members of the viral K3 family localize to intracellular membranes, mostly the ER. It is therefore unclear at what stage of intracellular transport target glycoproteins become ubiquitinated. It is possible that ubiquitination occurs en route to the cell surface or target proteins could be ubiquitinated during recycling. The localization of MARCH-IV and -IX in the Golgi and trans-Golgi network, respectively, is consistent with either possibility.
The MARCH family represents a novel family of ubiquitin ligases with a noncanonical RING domain for which we adopted the name RING-CH-domain, as previously proposed for the SSM4/DOA10 protein (35
). A close relationship between the RING-CH domain and other RING domains is also suggested by structural predictions, whereas other PHDs are predicted to fold into structures that are clearly distinct from the RING domain (2
). The RING-CH domain thus expands the RING-E3 family, already the most abundant E3 family, with potentially several hundred members (26
). In contrast to the MARCH family, however, most RING-E3s are either cytosolic, nuclear, or peripheral membrane proteins (26
). Two examples for transmembrane RING-E3s that are not MARCH proteins are Der3/Hrd1 (7
) and p78 (12
). These E3s have all been shown to be involved in the degradation of misfolded proteins in the ER or ER-associated protein degradation. MARCH-VI, the human homologue of DOA10/SSM4, is also likely to be involved in ER-associated protein degradation. In contrast, MARCH-IV and -IX (the present study) and MARCH-VIII (16
) direct the internalization of their substrates. It seems likely that endocytosis, rather than proteasomal degradation, will also be the mechanism of the other two transmembrane MARCH proteins, since most of them seem to locate to the endosomal-lysosomal compartment.
The membrane association of MARCH proteins implies that their corresponding ubiquitin-conjugating enzymes are also membrane associated or need to be recruited to the membrane. Indeed, experimental evidence suggests that SSM4/DOA10, p78, and Der3/Hrd1 cooperate with ubc6 and ubc7 (7
). Ubc6 and ubc7 are bound to the ER membrane by a transmembrane domain and by interacting with another protein, respectively (4
). It is thus likely that MARCH-VI will interact with the human homologs of ubc6 or ubc7. MARCH proteins that leave the ER and regulate internalization, however, are more likely to interact with different E2s in vivo. Although the E2s interacting with MARCH proteins in vivo have yet to be identified, our in vitro ubiquitination experiments suggest that the amino-terminal regions of each MARCH protein dictate a preference for certain E2s.
At present we can only speculate about the physiological role of the MARCH proteins. The results obtained with forced expression of MARCH proteins in our experiments strongly suggest that several members of this family regulate the degradation of glycoproteins at the cell surface. MARCH-IV and MARCH-IX could be involved in the internalization of free heavy chains upon dissociation of peptide and β2
-microglobulin. However, gene knockdown of MARCH-IX by small interfering RNA did not affect the turnover of MHC heavy chains in HeLa cells (data not shown). Alternatively, it might be that MARCH-IV and -IX have to be upregulated by external stimuli before they affect MHC-I turnover, whereas they are involved in the turnover of other cell surface protein at basal levels. Since we only screened a small number of cell surface proteins, it is highly likely that MARCH proteins will be involved in the internalization of other, yet-to-be-identified transmembrane proteins. Many cell surface receptors are internalized upon engagement with ligands, either soluble or bound to the surface of neighboring cells (26
). The involvement of ubiquitin in these processes is just beginning to emerge and the regulation of ubiquitination is not known. We therefore speculate that MARCH proteins will be involved in regulating receptor internalization as well as protein turnover.
The functional relationship between MARCH proteins and the viral K3 protein family suggests that the viral proteins were derived from their cellular homologues. There is ample precedence for common host-related immunomodulators in both herpesviral and poxviral genomes (29
). These host-derived proteins, however, often assume a new or modified function, possibly even acting as inhibitors of their host counterparts. Whereas in most of these cases the known function of the host-protein served as a road map to elucidate the function of the viral protein, the reverse strategy has been used here to discover the function of a novel eukaryotic protein family.