In this study, we characterized human Surf4, and we found it to be associated with the ERGIC and to cycle in the early secretory pathway in a dilysine signal-dependent manner. Erv29p, the yeast orthologue of Surf4, acts as a cargo receptor for glycosylated α-factor in yeast (Belden and Barlowe, 2001
; Otte and Barlowe, 2004
). Although a knockdown of Surf4 had no effect on total protein secretion, it remains possible that human Surf4 also operates as a cargo receptor for a limited set of proteins that would not be apparent in a global secretion assay. Previous studies have also implicated Erv29p in ER quality control. In yeast cells lacking Erv29p, misfolded soluble proteins are stabilized, and it was proposed that efficient degradation of these misfolded proteins requires transport between ER and Golgi mediated by Erv29p (Caldwell et al., 2001
). We found no equivalent function for human Surf4. An efficient knockdown of Surf4 had no effect on the degradation of the Z mutant of α1-antitrypsin a prototype ERAD substrate (data not shown). This observation argues against a general role of Surf4 in ER degradation of misfolded soluble proteins as suggested for Erv29p.
The characterization of Surf4-interacting proteins uncovered a novel role of cargo receptors in maintaining the architecture of ERGIC and Golgi. Surf4 was found to form at least two protein complexes, one complex that has an Mr
of 232 kDa and comprises p23, p24, and p25; and another complex of ~60 kDa, which was not further characterized but may contain KDEL-receptor. The serendipitous finding of a coimmunoprecipitation of Surf4 and ERGIC-53 suggests the existence of a third complex. Because ERGIC-53 forms homohexamers (Schweizer et al., 1988
), this complex can be expected to be very large so that it may not have entered the Blue Native gel. It is widely recognized that p24 family proteins form heterooligomeric complexes with one another, which complicates the functional analysis of these proteins (Dominguez et al., 1998
). The current study suggests that the situation is even more complex. The major known cargo receptors can form various protein complexes with one another with functional implications for organelle maintenance. Although this was unexpected, an even greater surprise was the observation that a double knockdown of Surf4/ERGIC-53 and a single knockdown of p25 resulted in an identical Golgi and ERGIC phenotype, particularly because the Surf4/ERGIC-53 knockdown did not affect p25 levels and vice versa. There are no indications, however, for a major difference of the phenotypes resulting from the two different knockdowns, neither at the light nor at the ultrastructural level. The phenotype is characterized by a reduced number of ERGIC clusters and fragmentation of the Golgi apparatus whereby the Golgi elements were not randomly distributed in the cytoplasm but largely remained in the original area of the initially compact Golgi.
Numerous situations are known in which the Golgi assumes a fragmented phenotype. How do these phenotypes compare with that observed in the present study? The classical phenotype of dispersed Golgi is due to disruption of microtubules by microtubule-active drugs, such as nocodazole. By contrast, silencing of Surf4/ERGIC-53 or p25 had no effect on microtubules (unpublished data) and the Golgi mini-stacks were not randomly distributed in the cytoplasm as in nocodazole-treated cells. Some other knockdown conditions can lead to Golgi fragmentation similar to that described here, although effects on the ERGIC have not been studied. For example, silencing the SNARE protein syntaxin 5 results in Golgi fragmentation that barely affects anterograde transport of VSV-G, but the underlying mechanism is unknown (Suga et al., 2005
). Silencing of KAP3, the nonmotor subunit of kinesin 2, also results in fragmentation of the Golgi (Stauber et al., 2006
). Again, anterograde secretory traffic is unaffected, but KDEL-receptor–dependent retrograde transport is abrogated, presumably due to an unexplained redistribution of the KDEL-receptor to the ER. Thus, this phenotype is different. Yet another type of Golgi fragmentation results from silencing golgin-84 (Diao et al., 2003
). However, this phenotype is accompanied by changes of the ER, and it has been attributed to a defect in anterograde trafficking. Comparing all the known Golgi fragmentation phenotypes, the Golgi phenotype induced by cargo receptor silencing is strikingly similar to that recently reported for knockdowns of the Golgi matrix proteins GM130 and GRASP65 (Puthenveedu et al., 2006
). Either knockdown prevents lateral linking of Golgi stacks resulting in mini-stacks. GM130 mediates stabilization and targeting of GRASP65, and the two proteins are required for Golgi ribbon formation. As a further similarity to the current work, secretory transport is independent of GM130-mediated Golgi ribbon formation (Puthenveedu et al., 2006
). Importantly, however, there was no indication of dissociation of GM130 or GRASP65 in cargo receptor knockdowns in the current study, indicating that these two Golgi matrix proteins are not sufficient for Golgi ribbon formation. Moreover, a knockdown of GM130 has no effect on the stability of the ERGIC (our unpublished observations).
Reduced COP I binding for both knockdowns of Surf4/ERIGC-53 and p25 provided a mechanistic explanation for at least some aspects of the phenotype. There are two major different functions of COP I: vesicle formation and stabilization of membranes (Klausner et al., 1992
; Rothman, 1994
; Storrie, 2005
; Bethune et al., 2006a
). COP I vesicles mediate membrane traffic within the Golgi, from cis
-Golgi to ERGIC, and from ERGIC to ER. Some rapidly cycling transmembrane proteins are actively recruited to retrograde vesicles by a dilysine signal of their cytosolic tail that directly interacts with COP I subunits (Jackson et al., 1990
; Cosson and Letourneur, 1994
; Bethune et al., 2006a
). Surf4, ERGIC-53, and p25 contain such a dilysine signal that is functional in all three proteins (Itin et al., 1995
; Emery et al., 2003
; this study). In vitro, the formation of COP I vesicles requires the presence of the cytoplasmic domains of p24 family proteins (Bremser et al., 1999
). Thus, COP I dissociation from cis
-Golgi and ERGIC observed in the current study renders retrograde traffic less efficient. Because anterograde secretory traffic is unaffected this obviously leads to a shortage of ERGIC membranes, which would explain the reduced number and perhaps also shortened life span of ERGIC clusters. For such an outcome with reduced ERGIC-53 cluster numbers one would have to also postulate that in the knockdown cells ERGIC-to-ER transport, although reduced, is slightly more efficient than cis
-Golgi to ERGIC transport. This is plausible in view of the proximity of ERGIC and ER, but it cannot be assessed experimentally with current technology.
A function of COP I in membrane stabilization is known from experiments with BFA. On BFA treatment, COP I dissociates from Golgi membranes, and these membranes rapidly tubulate and fuse with the ER. Obviously, COP I protects membranes from tubulation and thereby guarantees organelle integrity and identity. Importantly, neither silencing Surf4/ERGIC-53 nor p25-induced Golgi tubulation despite considerable dissociation of COP I. Under these knockdown conditions COP I dissociation can be assumed to occur at the level of the ERGIC and cis
-Golgi, the recycling sites of these cargo receptors. In contrast, overexpression of p25 containing an inactivated dilysine signal does not affect COP I distribution or induce fragmentation of the Golgi apparatus, although it mislocalizes p24 family members to the cell surface (Emery et al., 2003
). Inversely, the depletion of p25 did not lead to mislocalization of endogenous p24 to the cell surface (unpublished data). Obviously, overexpression of mutated p25 does not impair the function of p25 to the same extent as a knockdown of p25.
Clearly, COP I dissociation induced by cargo receptor silencing does not result in a BFA-like effect. Thus, COP I depletion cannot explain the absence of tubulation of the cis
-Golgi. Together with the partial resistance of the cis
-Golgi to BFA after cargo receptor silencing, the lack of tubules implies that cargo receptors are required for efficient tubulation. A likely scenario is that cargo receptor tails mediate the interaction of cis
-Golgi membranes with microtubules. Microtubules are required for BFA-induced tubulation of Golgi membranes after COP I dissociation and their subsequent consumption by the ER (Lippincott-Schwartz et al., 1990
). Receptor tails may recruit kinesine-type motor proteins, such as kinesin II (Stauber et al., 2006
), in the absence of protective COP I coats. Consistent with such a mechanism, the tubulation of anterograde transport intermediates also depends on cargo receptor tails as microinjection of cytosolic tails of p23 and p24 efficiently inhibits tubule formation (Simpson et al., 2006
). Obviously, p24 and presumably other cargo receptor tails have an inherent tubulation potential which needs to be controlled by COP I coats to maintain Golgi integrity.
Is the Golgi fragmentation in Surf4/ERGIC-53 or p25 knockdown cells due to COP I dissociation? The close similarity of phenotypes resulting from matrix or cargo receptor knockdowns raises the question of whether an interaction of the two classes of proteins is required for maintaining the Golgi ribbon. If so, a knockdown of either protein class would cause an identical Golgi mini-stack phenotype. Such a notion is not entirely hypothetical because p23, p24, and p25 have been reported to be in a complex with GRASP65, GRASP55, and GM130 in vivo and purified GRASPs directly bind to cytoplasmic tails of p24s (Barr et al., 2001
). In contrast to these observations, we have not seen an interaction of p25, Surf4, or ERGIC-53 with GM130 in immunoprecipitation experiments with antibodies to GM130 (data not shown). Thus, more detailed studies will be required to assess a putative dual interaction of cargo receptors with COP I and matrix proteins. It is worth noting, however, that the ERGIC phenotype induced by cargo receptor silencing is unlikely to be due to impaired matrix/tail interactions, because GM130 is primarily associated with the first Golgi cisterna at steady state (Nakamura et al., 1995
; Taguchi et al., 2003
) and is not detectable in the ERGIC (). An alternative possibility to explain the Golgi phenotype induced by receptor silencing is a disturbed balance of the amount of Golgi membranes and matrix proteins. Reduced retrograde traffic from cis
-Golgi to ERGIC may result in an increase in Golgi membranes without a corresponding increase in matrix proteins, which may affect Golgi ribbon maintenance.
Why does a single knockdown of Surf4 or ERGIC-53 not change Golgi morphology, whereas p25 does? Currently, we can only speculate about the underlying mechanism. One possibility is that the individual levels of ERGIC-53 and Surf4 in the cis
-Golgi are lower than those of p25; therefore, only a combined knockdown of Surf4 and ERGIC-53 leads to sufficient dissociation of COP I from the cis
-Golgi. Although no information for Surf4 is available, the levels of ERGIC-53 in the cis
-Golgi are indeed low, because the recycling of ERGIC-53 between ERGIC and ER largely bypasses the cis
-Golgi (Klumperman et al., 1998
; Ben-Tekaya et al., 2005
). Alternatively, p25 may not act in isolation because it forms complexes with other p24 proteins that are known to interact with COP I coats via a diphenylalanine rather than a dilysine signal (Bethune et al., 2006a,b
). By indirectly affecting other p24 family members, silencing of p25 may have a greater impact.
In conclusion, we propose the following model for the changes of the early secretory pathway induced by the depletion of Surf4/ERGIC-53 or p25 (). The reduction of cargo receptor tails reduces COP I binding to cis-Golgi and ERGIC and impairs retrograde vesicular traffic. Because anterograde traffic is unchanged this defect results in fewer ERGIC clusters. The reduction of cargo receptors in the cis-Golgi also leads to Golgi mini-stacks either due to insufficient cross-linking of cargo receptor tails with Golgi matrix or due to an imbalance of Golgi membranes and Golgi matrix. According to the maturation model, mini-stack formation would start at the cis-Golgi and gradually be completed as the first cis-Golgi cisterna moves and matures in cis-to-trans direction. Whatever the precise mechanism, the current study shows that networks of established and putative cargo receptors are required to maintain the architecture of ERGIC and Golgi. Thus, cargo receptors of the early secretory pathway can have multiple functions by operating both individually and in concert with one another. This striking dual mode of operation will have to be taken into consideration in future attempts to understand the organization and function of the secretory pathway.
Figure 11. Model depicting the effect of silencing Surf4/ERGIC-53 or p25 on the early secretory pathway. In the presence of cargo receptors (+cargo receptors), the architecture of the organelles is guaranteed by balanced anterograde and retrograde trafficking indicated (more ...)