Rabs are intrinsically cytosolic proteins, yet only function when in association with membranes. The posttranslational modification of prenylation is a prerequisite for membrane attachment. In addition to membrane association, proper targeting of Rab proteins is essential for their function in regulating membrane traffic; a characteristic feature of Rab proteins is their steady-state localization to the cytosolic surface of a particular subcellular membrane. Pioneering experiments examining the relationship of membrane attachment and function led to the idea that prenylation is a convenient method of giving proteins the physiochemical ability to stably attach to lipid bilayers, enabling regulation through reversible recruitment onto membranes (Leevers et al., 1994
; Stokoe et al., 1994
). In the case of Rab proteins, this idea was underscored by the dramatic finding that the lipid tail could be circumvented by giving the Rab proteins a transmembrane domain, provided that the transmembrane domain took them to the correct location (Ossig et al., 1995
). Obviously, Rab-GDI–mediated recycling did not occur in this situation; however, this requirement could be eliminated by protein overexpression. In these experiments, the replacement of the lipid anchor with a transmembrane domain that targeted the Rab protein to the correct compartment in this experiment may have obscured any possible specific role played by the lipid anchor. The availability of genomic information, a better understanding of the enzymes involved in prenylation, and genetic models that can distinguish between different levels of function have allowed us to examine this question.
To explore the specific role of the lipid modification in Rab protein function, we asked whether a single lipid geranylgeranyl group could substitute for the two geranylgeranyl groups found on most Rab proteins, and, if so, could a shorter lipid group such as a farnesyl group substitute for the longer geranylgeranyl groups? We created Rab prenylation variants by replacing the double cysteine motif at their C terminus with CAAX boxes to study the localization and function of the singly prenylated Rab proteins. C-terminal CTIM or CIIL box versions of the essential Rab genes YPT1
were unable to function in vivo when expressed as the only copy in the cell. Although Rab-GDI plays a critical role in the membrane targeting and recycling of Rab proteins, it is also thought that the homologous protein REP can function in this manner. Because REP is the chaperone that presents the Rab protein to GGTaseII, it is thought that REP mediates the very first membrane-targeting event in the existence of the Rab protein. If this is the case, and if the REP-mediated targeting is critical, perhaps the CAAX box variants we constructed were unable to function correctly because these sequences are in vivo substrates for FTase and GGTaseI. To eliminate this possibility we created C-terminal variants that contained a single cysteine-to-serine point mutation of one of the residues that is prenylated by GGTaseII. It has been previously demonstrated that such mutants remain the substrates of a single round of prenylation by GGTaseII and so would exist as a complex with REP, which could then target them to membranes (Wilson et al., 1996
). Such mutants would be singly geranylgeranylated exclusively by GGTaseII in combination with REP so eliminating any contribution from GGTaseI. Our finding that even singly geranylgeranylated YPT1
variants that are the substrates of GGTaseII cannot function as the only copy in the cell indicates that it is the specific double prenylation modification that is required for full function. We did, however, uncover differences in the mono-geranylgeranylated proteins that result from different prenyltransferase enzymes. ypt1CIIL
, the substrates for either GGTaseI or GGTaseII, were able to suppress temperature-sensitive alleles ypt1-3
, while the exclusive GG-TaseII substrates ypt1C205S
were not. These data agree with previous studies demonstrating that Rab proteins mutated to GGTaseI substrate CAAX boxes can in fact support function, provided that sufficient Rab protein reaches the correct membrane (Soldati et al., 1993
; Overmeyer et al., 2001
). It is possible that the ypt1C205S
are not released from REP after a single round of prenylation, because REP has been reported to form a very tight, stable complex with mono-geranylgeranylated Rab protein (Thoma et al., 2001
), and this could explain differences observed between the two set of mutants.
Using Ypt1p and Sec4p as examples, we also investigated whether the prenylation variants we created are indeed modified by asking if they could still partition into the detergent phase of a Triton X-114 partition. Each of the prenyl variants was able to partition into the detergent phase, in contrast to an unprenylated ΔCC mutant (). These data suggest that the effects we observe in Rab protein functionality with these variants can be attributed to the alternative Rab prenylation. Although other lipid anchor sequences on Rab proteins receive lipid modifications, they do not lead to correct function.
Why do mono-prenylated Rab proteins fail to function? One possibility is that alternative lipid modifications fail to stably associate with membranes. This may well be the case for farnesylated proteins (C15 moiety). However, singly geranylgeranylated proteins, with their C20 lipid tails are more than two log(P) units more nonpolar than farnesyl groups (Black, 1992
). Geranylgeranylation significantly enhances the bilayer partitioning ability of the modified protein. Although mono-geranylgeranylated proteins have the biophysical ability to stably associate with membranes, our data indicate that they are nonfunctional because they are unable to localize to the correct subcellular compartment. In each case examined, Sec4p, Ypt1p, Ypt6p, Ypt7p, and Vps21p, the monoprenylated variants did not localize in the same manner as their wild-type equivalents. Moreover, in the case of Sec4p, untagged prenyl variants examined by indirect immunofluorescence, gave similar results (). These data suggest that for Rab proteins, lipid modification plays dual functions. It is required for both membrane association and localization or clustering; prenylation is necessary for the former, and di-geranylgeranylation is required for the latter.
How applicable are these results to Rab proteins in general? In this study, we have examined the functionality of two different Rab proteins and the localization of prenylation variants of five different Rab proteins to reach our conclusion that dual prenylation is specifically required for Rab protein function and localization. While preparing this article, we became aware of a similar study in mammalian cells that reached the same conclusions (Gomes et al., 2003
). We therefore believe that our results show a common principle of Rab protein function, namely, a specific requirement for double prenylation. The original impetus for the experiments we report in this study was the desire to create prenylated peptide constructs of Rab hypervariable sequences to examine the possibility that such constructs might act as dominant inhibitors of endogenous Rab membrane recruitment. We expected that singly geranylgeranylated Rab proteins would be indistinguishable from wild type and were surprised by our results that mono-prenylated Rab proteins were nonfunctional. However, double prenylation is a characteristic hallmark of the majority of Rab GTPase family members, a family that is conserved in all eukaryotes. In fact, it would be surprising that a group of proteins would evolve this specialized dual prenylation modification and the machinery to produce it without a biological imperative.
We examined known Rab-interacting factors for the possible existence of protein entities that recognize the specialized dual prenylation of Rab proteins. We confined our list to factors conserved from yeast to human that are known to require an intact C-terminal cysteine motif for productive Rab protein interactions. The results of these experiments lead us to propose the YIP1 family of proteins as potential candidates through which the di-geranylgeranylation specificity is mediated. Yip1p was originally identified as a factor specific for Ypt1p and Ypt31p interaction (Yang et al., 1998
). However, Sec4p is as homologous to Ypt1p and Ypt31p as either is to each other, and it has become appreciated recently that Yip1p is capable of pleiotropic Rab protein interactions (Matern et al., 2000
; Calero et al., 2002
), which we confirm in this study. Our data show that Yip1p can interact with the di-geranylgeranylated Rab proteins Ypt1p, Sec4p, Ypt31p, Vps21p, Ypt6p, Ypt7p, Ypt52p, and Ypt53p. Yip1p does not interact with mono-geranylated Sec4p proteins. It is also of note that several mammalian Rab proteins such as Rab8 contain CAAL motifs that are singly geranylgeranylated both by REP/GGTase II and by GGTaseI (Wilson et al., 1998
). We would predict that such proteins may be insensitive to the impact of YIP1-like family members and demonstrated that such proteins are unable to interact with human YIP1A, although, as we have demonstrated for Sec4p mutants with CAAX boxes, Rab-GDI can still bind these monoprenylated Rab proteins. It should be noted that Sec4p is more homologous in primary sequence to either Rab8 or Rab13 (49.3 and 52.2% identity, respectively) than to Ypt1p (44.4% identity), its closest homolog in yeast. The fact that both human YIP1A and yeast Yip1p are capable of interactions with Sec4p, Rab1a, Ypt1p, and Ypt31p, all di-geranylgeranylated members of the same Rab subfamily (Pereira-Leal and Seabra, 2000
), but not with mono-geranylgeranylated Rab8 or Rab13, leads us to conclude that it is the di-geranylgeranylation that is the critical factor for YIP1 interaction. The relevance of our findings showing cross-species protein interaction is reflected in our demonstration showing the conservation of YIP1 protein function. Human YIP1A can fully substitute for YIP1, an essential gene in yeast. Together with our data showing no interaction between Yip1p and mono-geranylgeranylated Sec4p variants (), these results show that di-geranylgeranylation is critical for interactions between Yip1p and Rab GTPases and additionally demonstrate that the interactions of Yip1p with Rab GTPases are well conserved in evolution. Due to our finding that the requirement of di-geranylgeranylation for Rab protein function correlates with specific Rab protein localization, we sought to examine whether Yip1p might play a role in Rab protein localization. Using the mutant allele, yip1-4
, we demonstrate that loss of functional Yip1p has an impact on the localization of Ypt1p, shifting it from Golgi localization to a diffuse pool. These results demonstrate that Yip1p can impact Ypt1p localization in vivo. Together with our results showing loss of localization of the mono-prenylated Rab proteins, and the failure of such mutants to interact with Yip1p, these data suggest that Yip1p and other YIP1-family members are candidates for factors through which di-geranylgeranylated Rab proteins work to achieve correct membrane localization. It should be noted, however, that in this study we only tested known Rab-interacting factors, and there may be additional proteins present in the proteome that also specifically recognize digeranylgeranylated Rabs and aid in their correct localization. YIP1
is an essential gene, and yip1-4
cannot be suppressed by overexpression of other YIP1 family members in yeast (our unpublished data). These data are surprising considering that an ability to promiscuously associate with dual prenylated Rab proteins is the only known function for YIP1-family proteins and suggest either that Yip1p contains additional unique functions or that it interacts with, and is responsible for, an essential Rab protein. Four members of the YIP1-protein family and 11 Rab proteins have been identified in yeast. YIP1-family members associate both among themselves as well as with other proteins (Matern et al., 2000
; Calero et al., 2001
; Calero and Collins, 2002
), and one possibility may be that a combinatorial assortment of YIP1 family complexes confer specificity toward different Rab proteins. In vivo, the accessibility of Yip1p to Rab proteins may be restricted by its localization and interacting partners.
In summary, our findings demonstrate a specific lipid requirement of double geranylgeranylation for the Rab GTPase class of proteins to function correctly and show that double geranylgeranyl groups are required for the Rab protein to localize to its characteristic organelle membrane. The exact mechanism by which the di-geranylgeranylated proteins act to achieve correct localization remains to be uncovered. Although different prenylation will affect the membrane-partitioning ability of the modified protein, isoprenylation may have an additional role and be recognized by another protein. Our data indicate the YIP1 family as possible effector candidates for the di-geranylgeranylated Rab proteins, although further work is needed to explore the biochemical basis and physiological relevance of the YIP1–Rab interactions.