(Received for publication, December 12, 1995; and in revised form, January 18, 1996)
From the
Synaptobrevin is a tail-anchored protein with a hydrophobic C-terminal transmembrane segment that inserts into the endoplasmic reticulum membrane independently of the SRP/Sec61p pathway. Here, we show that idealized hydrophobic segments composed of 11-17 leucines and 1 valine function as insertion signals in vitro, whereas shorter segments do not. These results suggest that there are no specific requirements beyond overall hydrophobicity for C-terminal endoplasmic reticulum insertion signals.
A class of cytoplasmically exposed membrane proteins with a
C-terminal membrane anchor (tail-anchored proteins) has recently
attracted attention since it encompasses members of the family of
soluble N-ethylmaleimide-sensitive factor attachment protein
receptor (SNARE) proteins. Other tail-anchored proteins are
ER()-bound enzymes such as cytochrome b
, heme oxygenase, and microsomal aldehyde
dehydrogenase, and the class also includes certain viral proteins such
as the middle T antigen(1) . In contrast to the majority of
integral membrane proteins, tail-anchored proteins have no N-terminal
signal sequence and do not utilize the SRP/Sec61p pathway for targeting
and membrane insertion(2, 3) .
Synaptobrevin/vesicle-associated membrane protein (VAMP) is a
tail-anchored SNARE protein functioning in the fusion of synaptic
vesicles to the plasma membrane in neuronal and neuroendocrine cells (4, 5, 6, 7) . Synaptobrevin first
inserts into the ER membrane and is then transported through the
secretory pathway to synaptic vesicles(2) . ER insertion is
post-translational and requires ATP and as yet unidentified protein
components of the ER membrane distinct from the components of the
SRP/Sec61p pathway(2) . Membrane anchoring is provided by a
membrane-spanning, C-terminal hydrophobic tail(2, 3) ,
and further transport to synaptic vesicles may be determined by a
predicted amphiphilic -helix in the cytoplasmic
domain(8) .
While the subcellular sorting of tail-anchored proteins has thus been studied to some extent, the initial membrane insertion event is poorly characterized. Here, we show that targeting and insertion of human synaptobrevin 2 (Syb2) into dog pancreas microsomes in vitro can be mediated by a C-terminal tail containing as little as 12 hydrophobic residues, and that the precise amino acid sequence of the hydrophobic anchor is unimportant for this step.
To make the Syb2-nL
series of constructs, the PstI-BamHI fragment from
Syb2-G13 in pGEM3Gl (2) was cloned into phage M13mp18
(Pharmacia). An NdeI restriction site was introduced by
site-specific mutagenesis according to the method of Kunkel (10, 11) around amino acid Val
of
Syb2-G13, changing the DNA sequence from GTTTACTTC to GCATATGTC (NdeI site underlined). This changes the amino acid sequence
from Val
-Tyr-Phe to Ala
-Tyr-Val. In order
to introduce the polyleucine tails, double-stranded oligonucleotides
were cloned between the BclI site and the new NdeI
site of Syb2-G13 in pGEM3
Gl. Thus, the natural transmembrane
segment M
IILGVICAIILIIIIAYV was replaced with
M
MIKKKKL
VQQQPYV. All mutants were confirmed by
DNA sequencing of M13 or plasmid DNA using T7 DNA polymerase.
Figure 1: Deletion analysis of the Syb2 C-terminal insertion signal. A, amino acid sequences for full-length Syb2 and deletions mutants. The general sequence of the Syb2-nL constructs is also shown (n denotes the number of leucine residues in the hydrophobic segment; the C-terminal glycosylation acceptor site is underlined). B, full-length Syb2 and three C-terminal deletion mutants, Syb2-98, -104, -110, were synthesized in vitro and post-translationally incubated at 0 °C or 30 °C in the absence or presence of microsomes. To test for membrane insertion, samples were submitted to flotation in an alkaline sucrose gradient. The floated material was analyzed by SDS-PAGE and fluorography.
Figure 2:
Twelve but not nine hydrophobic residues
are sufficient to anchor Syb2 in the ER membrane. A, Syb2-17L
(which has 17 leucines and 1 valine in the hydrophobic segment) was
translated in vitro either in the absence (lanes 1 and 5) or presence (lanes 2-4 and 6-8) of microsomes. Acceptor peptide (AP), a
competitive inhibitor of N-linked glycosylation, was present
during the incubation in lane 3, and a related, noninhibiting
peptide (NAP) was present in lane 4. After
translation, microsomes were digested with proteinase K (lane
7) or with proteinase K after solubilization with Triton X-100 (lane 8). The weak background bands just above and in the
position of band b in lanes 1 and 5 are not
related to Syb2 (data not shown). The upper band purifies with
the supernatant (C) and thus represents a cytoplasmic protein
made from an mRNA contaminating the reticulocyte lysate. B,
Syb2-17L (lanes 1 and 2), Syb2-14L (lanes 3 and 4), Syb2-11L (lanes 5 and 6),
Syb2-8L (lanes 7 and 8), and Syb2-5L (lanes 9 and 10) were translated in vitro either in the
absence (odd-numbered lanes) or presence (even-numbered
lanes) of microsomes. Band a is nonglycosylated Syb2, band b is glycosylated Syb2. C, microsomes incubated
with the various constructs were washed in NaCO
(pH 11.5) and sedimented. S = supernatant, P = pellet.
To ascertain whether these compositional differences relate to different requirements for ER targeting and membrane integration, a number of mutants were made in the Syb2 C-terminal tail.
As shown in Fig. 2A,
the construct with 17 leucines in the tail (Syb2-17L) gave rise to a
higher molecular weight form in the presence of microsomes (compare lanes 1 and 2). The formation of this species was
inhibited by a competing glycosylation acceptor tripeptide (N-benzoyl-Asn-Leu-Thr-N-methylamide; lane
3) but not by a related non-acceptor peptide (N-benzoyl-Asn-Leu-(allo)Thr-N-methylamide; lane
4). Finally, proteinase K digested Syb2-17L both in the absence
and presence of detergent (lanes 7 and 8),
demonstrating that the large N-terminal domain is exposed toward the
cytoplasmic side of the microsomes. Also, removal of the C-terminal
glycosylation site by an Asn Gln mutation resulted in a
nonglycosylated but membrane-inserted molecule (data not shown). Thus,
the shift in molecular weight observed in the presence of microsomes
can be attributed to Asn-linked glycosylation of the acceptor site
present near the C terminus and demonstrates that Syb2-17L is inserted
in the correct transmembrane orientation. We conclude that a C-terminal
hydrophobic stretch composed of 17 leucines and 1 valine is sufficient
for targeting to and transmembrane integration of Syb2 in the
microsomal membrane.
Similar experiments were carried out for the other constructs with progressively shorter hydrophobic segments. Syb2-17L and Syb2-14L were glycosylated to about 33%, Syb2-11L to about 10%, and Syb2-8L and Syb2-5L not at all (Fig. 2B). Membrane anchoring was also assayed by alkaline extraction(16) . Only the glycosylated forms of Syb2-17L, Syb2-14L, and Syb2-11L were found in the membrane pellet (Fig. 2C). Thus, a 12-residue-long hydrophobic stretch (11 leucines and one valine) is just enough to provide proper targeting and transmembrane anchoring of Syb2.
Synaptobrevin belongs to a small group of tail-anchored integral membrane proteins and inserts into the ER membrane independently of the SRP/Sec61p machinery(2) . Compared to membrane-spanning hydrophobic segments in proteins that use the SRP/Sec61p machinery for membrane insertion, transmembrane segments in tail-anchored proteins are on the average shorter and have a distinct overall amino acid composition with less leucine and alanine (Table 1), suggesting specific sequence constraints that may relate to ER targeting and integration, to sorting events later in the secretory pathway, or to other functional requirements.
We have studied the first step in the synaptobrevin assembly pathway, i.e. targeting and insertion into the ER membrane. Our results show that there are no specific sequence requirements for this step: hydrophobic segments composed almost exclusively of leucine residues function as insertion sequences and form transmembrane anchors, provided that they are longer than about 12 residues. Interestingly, this apparent ``minimum length'' is significantly longer than what has been shown to be required for signal peptide and stop-transfer functions in the SRP/Sec61p pathway, where in both cases only 7-8 consecutive leucines are necessary(17, 18) .
Although the atypical amino acid composition of tail-anchored proteins thus cannot be explained by the requirements of ER integration per se, it may nevertheless reflect the fact that tail-anchored proteins need to avoid early mis-sorting to other abundant membrane systems such as mitochondria(19, 20) . It is also possible that some property of the transmembrane anchor may be important for retention in the proper compartment of the secretory pathway(21, 22) , although a signal for targeting to synaptic vesicles has recently been proposed to be located in the cytoplasmic domain of synaptobrevin(8) .