BACKGROUND
the identification of sequence motifs that direct proteins to appropriate intracellular and plasma membrane locales represents a fundamental objective of cell biology  <cit> .
once defined, such signals permit the identification of mechanisms underlying protein trafficking  <cit>  and the targeted delivery of therapeutic agents to those subcellular domains where they are most effective  <cit> .
unfortunately, the identification of trafficking signals is not trivial as they may consist of non-contiguous amino acids, whose juxta-position to form a binding site may only be apparent in the fully folded protein  <cit> .
even in cases where trafficking signals are comprised of contiguous amino acids, they may be hard to identify since they lie within discrete subunits of complex oligomeric proteins  <cit> .
moreover, trafficking signals may overlap  <cit> , act hierarchically  <cit> , or be capable of being switched from inactive to active states through second messenger regulation  <cit> .
a well-established strategy to identify trafficking signals has been to infer their necessity and sufficiency from their ability to direct simpler reporter proteins to which they are fused, to the appropriate cellular compartment  <cit> .
unfortunately, available reporter proteins have numerous limitations including their endogenous expression in diverse cells and tissues, lack of convenient cloning sites and potential interactions with intra- or extracellular proteins that may affect their subcellular distributions  <cit> .
even more significant is the limited capacity for detection of presently available reporters, not least because of their reliance upon the limited range of antibodies that are commercially available and the indirect nature of the detection methodology.
to circumvent such problems, we have engineered an alternative protein trafficking reporter system  consisting of a simple artificial type i integral protein bearing permissive intra- and extracellular multiple cloning sites, a green fluorescent protein moiety  that permits direct localisation by fluorescence imaging in live cells and epitope tags for biochemical detection of protein turnover and surface expression.
RESULTS
vector construction
to generate a suitable trafficking reporter , a region encoding egfp and unique extracellular restriction sites from pegfp-c <dig>  was first inserted into the cell surface expression vector, pdisplay,  to generate an intermediate egfp-pdisplay construct .
the entire protein coding region of egfp-pdisplay was then pcr amplified and cloned into a pegfp backbone lacking the egfp insert, thereby introducing a second, distinct, intracellular multiple cloning site .
the primary homologue, described henceforth as pin-g, is a 5042bp puc-based vector  containing all the desired protein coding elements in the correct reading frame .
properties of pin-g
transfection of pin-g into hek <dig> cells, and subsequent immunoblotting with antibodies against egfp and ha disclosed a novel  <dig>  kda band, which was absent from mock-transfected cells .
constructs encoding pin-g fused to a tgn targeting motif and the carboxy terminus of the voltage-gated potassium channel kv <dig>  gave bands of  and , respectively.
fluorescence images of pin-g-transfected hek <dig> cells  revealed strong green heterogeneous fluorescence within intracellular organellar structures and at the plasma membrane.
in contrast, egfp alone  was expressed homogeneously throughout the cell.
thus, the presence of an igκ leader sequence and the pdgf receptor transmembrane region, but no added trafficking motifs, appeared to direct pin-g via the secretory pathway to the cell surface.
to confirm this, first, the surface expression of pin-g was tested by immunostaining non-permeabilized fixed cells with anti-egfp  and anti-ha , and subsequent detection with cy3-labeled secondary antibodies.
in each case, cy- <dig>  fluorescence overlapped strongly with pin-g fluorescence at the cell margin, and was absent upon omission of primary antibody to mock-transfected cells .
next, the intracellular disposition of pin-g was resolved through immunocytochemistry with established antibody markers  to membranous organelles  including  the golgi apparatus   <cit> ,  early endosomes   <cit> ,  the endoplasmic reticulum   <cit>  and  the lysosomes  <cit> .
throughout, we found no evidence for cytotoxicity or inclusion body formation.
pin-g is a faithful trafficking reporter
next, we sought to establish the potential of pin-g as a reporter of protein trafficking by exploiting the observation that a contiguous carboxy terminal motif within tgn- <dig> is both necessary and sufficient to localise this protein to the trans-golgi network  <cit> .
thus, cdna encoding the tgn- <dig> signal with a corresponding stop codon, was cloned into the putative intracellular multiple cloning site of pin-g  to yield a construct, designated as pin-tgn.
fluorescence imaging of hek <dig> cells transfected with pin-tgn , revealed a high concentration of fluorescence in vesicles and membranes distributed proximal to the nucleus in a characteristic 'signet ring' pattern  distinct from that seen with either pin-g  or egfp .
further immunocytochemistry showed extensive co-localisation of pin-tgn with the golgi apparatus, but not markers of early endosomes, the er or lysosomes .
analysis of pin-g surface expression levels
to confirm the cell surface disposition of pin-g and pin-fusion constructs, and its facile resolution by cell sorting assays, facs analysis was used.
in mock-transfected cells , or those treated with cy <dig> secondary antibodies alone , most cells showed low fluorescence  and r <dig> ).
however, upon transfection with pin-g or pin-tgn, a new population of cells was revealed that showed strong fluorescence in the green channel, .
after surface labelingof pin-g and pin-tgn transfected cells, with anti-ha and cy <dig> primary and secondary antibodies, a novel cell population displaying both red and green fluorescence  emerged, corresponding to those expressing construct at the cell surface.
by defining f as the fraction of transfected cells in the r <dig> quadrant ), pin-g surface expression was much greater than that of pin-tgn .
a pin-g fusion protein identifies internalization determinants in the kv <dig>  carboxy terminus
available evidence suggests that kv <dig> family potassium channels contain one or more carboxy terminal internalization determinants  <cit> .
to identify such determinants, a construct was generated encoding pin-g to which the entire carboxy terminus of rat kv <dig>   had been fused .
in transfected hek <dig> cells, pin-jhe <dig> fluorescence was localized to intracellular membrane vesicles and the cell surface .
surface expression of pin-jhe <dig> was confirmed through immunocytochemistry of non-permeabilised cells treated at 4°c with antibodies raised against the ha epitope tag .
however, in contrast to pin-g , pin-jhe <dig> surface labeling appeared less intense as indicated by a decrease in green:red  surface staining .
since, pin-jhe <dig> contains putative internalization motifs, we questioned whether such diminished surface labeling at steady-state compared to pin-g, might reflect partial redistribution of pin-jhe <dig> into the endocytic pathway.
to test this notion, we determined the fate of surface-labeled pin-jhe <dig> compared to pin-g following a pulse chase-protocol with anti-ha antibody .
after  <dig> h of internalization prior to labeling with cy3-conjugated secondary antibodies , cells expressing pin-jhe <dig> showed diminished surface labeling compared to cells fixed and labeled immediately after anti-ha application i.e.
at time  <dig> h .
in contrast, a similar high level of surface labeling was seen at both t =  <dig> h and  <dig> h for cells expressing pin-g .
since tgn- <dig> is routed via the surface to the tgn  <cit> , we also tested internalization of anti-ha in cells expressing pin-tgn.
as shown in figs.
6b panels g and h, surface labeling of pin-tgn, while weak at t =  <dig> h, was negligible after  <dig> h.
inspection of the c-terminal sequence of kv <dig>  reveals the presence of several candidate internalization motifs .
in anti-ha pulse-chase experiments, a di-leucine mutant, leu594ala , surprisingly, showed enhanced intracellular and diminished cell surface cy3-labeling compared to pin-jhe <dig> .
in contrast, a tyr612-phe  mutant showed enhanced surface and reduced intracellular labeling .
facs analysis of cells expressing pin-jhe <dig> mutants at t =  <dig> and  <dig> hr yielded internalization half-lives commensurate with the imaging data of 160mins,  <dig> mins and infinity for pin-jhe811; l-a and y-f, respectively.
thus, like the homologous residue  in kv <dig> , tyrosine  <dig> serves as a critical internalization signal  <cit> .
discussion
in this paper, we describe a novel construct – pin-g – for the facile detection of trafficking determinants within membrane proteins.
expression of pin-g yields an artificial, gfp-tagged, protein – pin-g – whose characteristics and topology conform to the type i protein depicted in figure 1b.
thus, pin-g is recognised by surface-applied antibodies corresponding to each extracellular moiety and is also re-distributed by sequences appended to the putative intracellular domain.
the presence of pin-g at the cell surface  also suggests that it does not contain any intrinsic targeting or trafficking determinants.
however, the addition of sequences containing known or putative targeting determinants re-specifies the trafficking fate of the expressed fusion construct indicating that pin-g is a faithful reporter for visualising and analysing trafficking mechanisms.
equally salient is the utility of the pin-g system in identifying and dissecting motifs, such as the previously undocumented tyr <dig> in kv <dig> , that control the dynamics and expression of cell surface proteins, especially when, like kv <dig> , they have no specific radioactive ligands or where biotinylation strategies may not be straightforward.
in contrast to existing reporters  <cit> , the pin vector is a considerably more versatile tool, not least because of the presence of intra- and extracellular multiple cloning sites, epitope and gfp tags and lack of endogenous expression.
moreover, unlike with present reporters, any aberrant expression of pin-g or its fusion constructs , while absent in the present study, should be readily visualised.
such versatility could be extended even further to include the dynamic visualisation of interacting proteins in live cells  <cit> , and the development of combinatorial library approaches for identifying intracellular or extracellular signalling mechanisms affecting trafficking  <cit> .
other potential applications of pin technology include the generation of constructs with alternate fluorophores to gfp such as mrfp  <cit> , thereby, permitting the development of biosensors or energy transfer assays between pin-fusion constructs.
finally, the reasonably small size and simplicity of the pin construct, especially when compared to most integral proteins mediating signal transduction, should facilitate the analysis of trafficking in cells such as neurons which are hard to transfect  <cit>  and also the development of viral gene delivery methodologies where packaging size is critical  <cit> .
CONCLUSIONS
here we have described the construction and faithful expression of a novel trafficking reporter protein, specifically designed for the detection and characterization of autonomous trafficking motifs.
by incorporating epitope and gfp tags into a simple type i transmembrane protein, our construct permits the direct visualization and biochemical dissection of protein signals and signalling domains transplanted from much more complex parent membrane proteins.
the potential uses of the pin-g reporter are, however, not limited to trafficking studies and could serve as a platform for the development of novel biosensors and the high-throughput screening of protein-protein interactions.

 the identification of sequence motifs that direct proteins to appropriate intracellular and plasma membrane locales represents a fundamental objective of cell biology. once defined, such signals permit the identification of mechanisms underlying protein trafficking and the targeted delivery of therapeutic agents to those subcellular domains where they are most effective. unfortunately, the identification of trafficking signals is not trivial as they may consist of non-contiguous amino acids, whose juxta-position to form a binding site may only be apparent in the fully folded protein. even in cases where trafficking signals are comprised of contiguous amino acids, they may be hard to identify since they lie within discrete subunits of complex oligomeric proteins. moreover, trafficking signals may overlap, act hierarchically, or be capable of being switched from inactive to active states through second messenger regulation. a well-established strategy to identify trafficking signals has been to infer their necessity and sufficiency from their ability to direct simpler reporter proteins to which they are fused, to the appropriate cellular compartment. unfortunately, available reporter proteins have numerous limitations including their endogenous expression in diverse cells and tissues, lack of convenient cloning sites and potential interactions with intra- or extracellular proteins that may affect their subcellular distributions. even more significant is the limited capacity for detection of presently available reporters, not least because of their reliance upon the limited range of antibodies that are commercially available and the indirect nature of the detection methodology. to circumvent such problems, we have engineered an alternative protein trafficking reporter system consisting of a simple artificial type i integral protein bearing permissive intra- and extracellular multiple cloning sites, a green fluorescent protein moiety that permits direct localisation by fluorescence imaging in live cells and epitope tags for biochemical detection of protein turnover and surface expression.  vector construction to generate a suitable trafficking reporter, a region encoding egfp and unique extracellular restriction sites from pegfp-c was first inserted into the cell surface expression vector, pdisplay, to generate an intermediate egfp-pdisplay construct. the entire protein coding region of egfp-pdisplay was then pcr amplified and cloned into a pegfp backbone lacking the egfp insert, thereby introducing a second, distinct, intracellular multiple cloning site. the primary homologue, described henceforth as pin-g, is a 5042bp puc-based vector containing all the desired protein coding elements in the correct reading frame. properties of pin-g transfection of pin-g into hek cells, and subsequent immunoblotting with antibodies against egfp and ha disclosed a novel kda band, which was absent from mock-transfected cells. constructs encoding pin-g fused to a tgn targeting motif and the carboxy terminus of the voltage-gated potassium channel kv gave bands of and, respectively. fluorescence images of pin-g-transfected hek cells revealed strong green heterogeneous fluorescence within intracellular organellar structures and at the plasma membrane. in contrast, egfp alone was expressed homogeneously throughout the cell. thus, the presence of an igκ leader sequence and the pdgf receptor transmembrane region, but no added trafficking motifs, appeared to direct pin-g via the secretory pathway to the cell surface. to confirm this, first, the surface expression of pin-g was tested by immunostaining non-permeabilized fixed cells with anti-egfp and anti-ha, and subsequent detection with cy3-labeled secondary antibodies. in each case, cy- fluorescence overlapped strongly with pin-g fluorescence at the cell margin, and was absent upon omission of primary antibody to mock-transfected cells. next, the intracellular disposition of pin-g was resolved through immunocytochemistry with established antibody markers to membranous organelles including the golgi apparatus, early endosomes, the endoplasmic reticulum and the lysosomes. throughout, we found no evidence for cytotoxicity or inclusion body formation. pin-g is a faithful trafficking reporter next, we sought to establish the potential of pin-g as a reporter of protein trafficking by exploiting the observation that a contiguous carboxy terminal motif within tgn- is both necessary and sufficient to localise this protein to the trans-golgi network. thus, cdna encoding the tgn- signal with a corresponding stop codon, was cloned into the putative intracellular multiple cloning site of pin-g to yield a construct, designated as pin-tgn. fluorescence imaging of hek cells transfected with pin-tgn, revealed a high concentration of fluorescence in vesicles and membranes distributed proximal to the nucleus in a characteristic'signet ring' pattern distinct from that seen with either pin-g or egfp. further immunocytochemistry showed extensive co-localisation of pin-tgn with the golgi apparatus, but not markers of early endosomes, the er or lysosomes. analysis of pin-g surface expression levels to confirm the cell surface disposition of pin-g and pin-fusion constructs, and its facile resolution by cell sorting assays, facs analysis was used. in mock-transfected cells, or those treated with cy secondary antibodies alone, most cells showed low fluorescence and r ). however, upon transfection with pin-g or pin-tgn, a new population of cells was revealed that showed strong fluorescence in the green channel,. after surface labelingof pin-g and pin-tgn transfected cells, with anti-ha and cy primary and secondary antibodies, a novel cell population displaying both red and green fluorescence emerged, corresponding to those expressing construct at the cell surface. by defining f as the fraction of transfected cells in the r quadrant ), pin-g surface expression was much greater than that of pin-tgn. a pin-g fusion protein identifies internalization determinants in the kv carboxy terminus available evidence suggests that kv family potassium channels contain one or more carboxy terminal internalization determinants. to identify such determinants, a construct was generated encoding pin-g to which the entire carboxy terminus of rat kv had been fused. in transfected hek cells, pin-jhe fluorescence was localized to intracellular membrane vesicles and the cell surface. surface expression of pin-jhe was confirmed through immunocytochemistry of non-permeabilised cells treated at 4°c with antibodies raised against the ha epitope tag. however, in contrast to pin-g, pin-jhe surface labeling appeared less intense as indicated by a decrease in green:red surface staining. since, pin-jhe contains putative internalization motifs, we questioned whether such diminished surface labeling at steady-state compared to pin-g, might reflect partial redistribution of pin-jhe into the endocytic pathway. to test this notion, we determined the fate of surface-labeled pin-jhe compared to pin-g following a pulse chase-protocol with anti-ha antibody. after h of internalization prior to labeling with cy3-conjugated secondary antibodies, cells expressing pin-jhe showed diminished surface labeling compared to cells fixed and labeled immediately after anti-ha application i.e. at time h. in contrast, a similar high level of surface labeling was seen at both t = h and h for cells expressing pin-g. since tgn- is routed via the surface to the tgn, we also tested internalization of anti-ha in cells expressing pin-tgn. as shown in figs. 6b panels g and h, surface labeling of pin-tgn, while weak at t = h, was negligible after h. inspection of the c-terminal sequence of kv reveals the presence of several candidate internalization motifs. in anti-ha pulse-chase experiments, a di-leucine mutant, leu594ala, surprisingly, showed enhanced intracellular and diminished cell surface cy3-labeling compared to pin-jhe. in contrast, a tyr612-phe mutant showed enhanced surface and reduced intracellular labeling. facs analysis of cells expressing pin-jhe mutants at t = and hr yielded internalization half-lives commensurate with the imaging data of 160mins, mins and infinity for pin-jhe811; l-a and y-f, respectively. thus, like the homologous residue in kv, tyrosine serves as a critical internalization signal. discussion in this paper, we describe a novel construct – pin-g – for the facile detection of trafficking determinants within membrane proteins. expression of pin-g yields an artificial, gfp-tagged, protein – pin-g – whose characteristics and topology conform to the type i protein depicted in figure 1b. thus, pin-g is recognised by surface-applied antibodies corresponding to each extracellular moiety and is also re-distributed by sequences appended to the putative intracellular domain. the presence of pin-g at the cell surface also suggests that it does not contain any intrinsic targeting or trafficking determinants. however, the addition of sequences containing known or putative targeting determinants re-specifies the trafficking fate of the expressed fusion construct indicating that pin-g is a faithful reporter for visualising and analysing trafficking mechanisms. equally salient is the utility of the pin-g system in identifying and dissecting motifs, such as the previously undocumented tyr in kv, that control the dynamics and expression of cell surface proteins, especially when, like kv, they have no specific radioactive ligands or where biotinylation strategies may not be straightforward. in contrast to existing reporters, the pin vector is a considerably more versatile tool, not least because of the presence of intra- and extracellular multiple cloning sites, epitope and gfp tags and lack of endogenous expression. moreover, unlike with present reporters, any aberrant expression of pin-g or its fusion constructs, while absent in the present study, should be readily visualised. such versatility could be extended even further to include the dynamic visualisation of interacting proteins in live cells, and the development of combinatorial library approaches for identifying intracellular or extracellular signalling mechanisms affecting trafficking. other potential applications of pin technology include the generation of constructs with alternate fluorophores to gfp such as mrfp, thereby, permitting the development of biosensors or energy transfer assays between pin-fusion constructs. finally, the reasonably small size and simplicity of the pin construct, especially when compared to most integral proteins mediating signal transduction, should facilitate the analysis of trafficking in cells such as neurons which are hard to transfect and also the development of viral gene delivery methodologies where packaging size is critical.  here we have described the construction and faithful expression of a novel trafficking reporter protein, specifically designed for the detection and characterization of autonomous trafficking motifs. by incorporating epitope and gfp tags into a simple type i transmembrane protein, our construct permits the direct visualization and biochemical dissection of protein signals and signalling domains transplanted from much more complex parent membrane proteins. the potential uses of the pin-g reporter are, however, not limited to trafficking studies and could serve as a platform for the development of novel biosensors and the high-throughput screening of protein-protein interactions. 
however, present reporters are limited by their endogenous expression, paucity of cloning sites, and difficult detection in live cells. powerful strategies to test candidate motifs include their ability to direct simpler reporter proteins, to which they are fused, to the appropriate cellular compartment. the identification of protein trafficking signals, and their interacting mechanisms, is a fundamental objective of modern biology. fluorescence imaging, flow cytometry and biochemical assays of transfected hek cells, confirm the size, topology and surface expression of pin-g. moreover, a pin-g fusion construct, containing a trans-golgi network targeting determinant, internalises rapidly from the cell surface and localises to the tgn. consequently, we have engineered a mammalian expression vector encoding a novel trafficking reporter – pin-g – consisting of a simple, type i integral protein bearing permissive intra/extracellular cloning sites, green fluorescent protein, cmyc and ha epitope tags. together, these data indicate that pin-g is a versatile, powerful, new reporter for analysing signals controlling membrane protein trafficking, surface expression and dynamics.
 the identification of protein trafficking signals, and their interacting mechanisms, is a fundamental objective of modern biology. unfortunately, the analysis of trafficking signals is complicated by their topography, hierarchical nature and regulation. powerful strategies to test candidate motifs include their ability to direct simpler reporter proteins, to which they are fused, to the appropriate cellular compartment. however, present reporters are limited by their endogenous expression, paucity of cloning sites, and difficult detection in live cells.  consequently, we have engineered a mammalian expression vector encoding a novel trafficking reporter – pin-g – consisting of a simple, type i integral protein bearing permissive intra/extracellular cloning sites, green fluorescent protein, cmyc and ha epitope tags. fluorescence imaging, flow cytometry and biochemical assays of transfected hek cells, confirm the size, topology and surface expression of pin-g. moreover, a pin-g fusion construct, containing a trans-golgi network targeting determinant, internalises rapidly from the cell surface and localises to the tgn. additionally, another pin-g fusion protein and its mutants reveal trafficking determinants in the cytoplasmic carboxy terminus of kv voltage-gated potassium channels.  together, these data indicate that pin-g is a versatile, powerful, new reporter for analysing signals controlling membrane protein trafficking, surface expression and dynamics. 