1. Substrate Specificity of Rhomboid Intramembrane Proteases Is Governed by Helix- Breaking Residues in the Substrate Transmembrane Domain 
Sinisa Urban, Matthew Freeman 
Molecular Cell 
Volume 11, Issue 6, Pages (June 2003)
DOI: /S (03)

2. Figure 1 Rhomboid-1 Displays Substrate Specificity
(A) Rhomboid-1 could cleave Spitz, but not EGFR, Delta, TGN38, or TGFα. The cleavage and subsequent secretion of each protein was assayed by Western blot of conditioned media from transiently transfected COS cells. These proteins could be detected in media due to the action of endogenous metalloproteases in the absence of the inhibitor Batimastat (MP). In addition to being absent in media, Rhomboid-1-cleaved forms of these proteins could not be detected in cell lysates (data not shown, but see Figure 3 for TGFα and Figure 4 for Delta).
(B) Cleavage of Spitz in the ER by Rhomboid-1-KDEL was examined in the presence or absence of Star. Rhomboid-1-KDAS served as a negative control, as it bears a mutated KDEL sequence and thus remained in the Golgi apparatus (Urban et al., 2001). Note that cleavage by Rhomboid-1-KDAS occurs in the Golgi apparatus and Spitz is rapidly secreted, while cleavage by Rhomboid-1-KDEL occurs in the ER and the cleaved form is not secreted (Urban et al., 2002a). The presence of Star had no effect on the cleavage of Spitz in the ER (the cleaved form is marked with an arrow). The positions of the molecular weight standards (in kDa) are depicted to the right of each panel.
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2
The Extracellular and Cytoplasmic Domains of Spitz Have No Role in Substrate Recognition by Rhomboid-1
(A) Schematic representation of chimeras with Spitz in black, TGFα in white, and the membrane bilayer represented by two horizontal lines (extracellular/luminal is up). The N terminus of Spitz was replaced with that of TGFα in a nested series at the last cysteine (residue 117) of the Spitz EGF domain (N117+C), at the end of the EGF domain (N123+C), at four residues before the TMD (N133+C), or just inside the TMD (N139+C) and compared to a control chimera (C) comprising the N-terminal region of Spitz. Each chimera also contained the C terminus of TGFα, which conferred a Star-independent localization throughout the secretory pathway. All proteins were detected by Western blot following expression in COS cells without Star and in the presence of 10 μM Batimastat. The cleaved product was detected in media and cells (arrow) in the presence of Rhomboid-1. Note that 3-fold more of the C chimera lysate sample was loaded, as it was expressed at lower levels than those containing the TGFα N terminus.
(B) The above chimeras were truncated within their TMDs to assess their relative secretion efficiencies in the absence of Rhomboid-1 cleavage; the N117 chimera was secreted more efficiently than the others.
(C) Spitz containing the C-terminal tail of TGFα (C) was cleaved as efficiently as wild-type Spitz (wt) in the presence of both Star (S) and Rhomboid-1 (R1) (upper panel shows the cleaved form in media), and in cells by Rhomboid-1-KDEL (lower panel). The full-length but not the cleaved forms (arrow) of the Spi-TGFα-C chimera are smaller than Spitz since the TGFα cytoplasmic tail is short.
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3
Rhomboid-1 Recognizes Spitz by a Small Motif in the Top Quarter of the Spitz TMD
(A and B) Replacing the Spitz TMD sequentially with that of TGFα (A) or TGN38 (B) did not decrease cleavage until the top quarter of the TMD (TMD-4/4) was replaced. The chimeras were assayed in the absence of Batimastat and detected by anti-GFP Western blot. Note that when cleavage failed in (A), the cleaved band (black arrow) was absent in media and cells, and in (A) and (B) the uncleaved Spitz accumulated as a hyperglycosylated full-length product in cells (white arrow).
(C) The Spitz substrate motif was sufficient to confer cleavage on TGFα by Rhomboid-1: a cleaved band was evident both in cells (arrow) and media for TGFα when it contained only the Spitz substrate motif (SM) but not for TGFα alone. All cleavage assays were performed in the presence of 10 μM Batimastat, except those marked MP (to indicate metalloprotease activity), and without Star.
(D) Sequence of the Spitz and TGFα TMDs, with the 7 residue substrate motif highlighted in white lettering.
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4
The Spitz Substrate Motif Is Sufficient for Rhomboid-1 Cleavage
(A) The Spitz substrate motif (SM) was engineered into the TMD of Delta by itself or with the Spitz juxtamembrane residues (SM+J). The presence of the Spitz SM was sufficient for cleavage by Rhomboid-1; a cleaved and secreted product was evident in the media fraction, and a cleaved band was present in cells (arrow).
(B) Cleavage of the Delta chimeras by Rhomboid-1-KDEL in the ER allowed a more direct comparison of the effect of the juxtamembrane domain on cleavage. Limiting the amount of Rhomboid-1-KDEL by reducing the amount of transfected DNA (in ng) revealed that both the SM and SM+J chimeras were cleaved at very similar efficiencies, while Delta+J only was not cleaved. The cleaved band was the same size as a Delta+SM chimera truncated in its TMD (sDl).
(C) Expression of Delta chimeras truncated in their TMDs revealed that the N-terminal domain of Delta+SM+J (Dl+J) was secreted more efficiently than that of Delta+SM. Wild-type Delta was secreted efficiently in the absence of Batimastat as a metalloprotease-cleaved form.
(D) Cleavage of a synthetic substrate containing GFP as its N terminus, the Spitz SM, and the C terminus of TGFα (SYN+SM) was compared to that of TGFα with the Spitz SM. The Rhomboid-1-cleaved SYN+SM band in media was always larger than the MP-cleaved form, while no SYN+SM cleaved form was detected in cells (as with other substrates including Spitz). Cleavage assays were performed in the presence of 20 μM Batimastat, but its absence (MP lanes) served as a positive control for the expression, cleavage by metalloproteases, and secretion of the substrates.
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5 Spitz Substrate Motif Determinants
(A) The Spitz substrate motif residues were individually placed into the TMD-4/4 chimera that contained the TGFα TMD and thus was not cleaved. Of the four residues unique to the cleaved TMD-3/4 chimera versus the uncleaved TMD-4/4 chimera, restoring only G143 converted the TMD-4/4 chimera into a Rhomboid-1 substrate, while restoring G143+A144 together resulted in cleavage that was as efficient as that of the TMD-3/4 chimera. Note that in addition to the cleaved product in the media, the hyperglycosylated uncleaved form in cells (white arrow) was decreased for the chimera containing the GA motif.
(B) The GA motif was introduced 2 residues up, or 1, 2, 3, 5, and 7 residues further into the TGFα TMD of the 4/4-TMD chimera to assess whether its position was constrained. GA chimeras in (A) and (B) were expressed at equivalent levels in cells, and cleavage was performed in the presence of 10 μM Batimastat.
(C) The intracellular cleavage of Spitz (arrow) was dependent on Rhomboid-1-KDEL levels (amount of Rhomboid DNA in ng). A141T consistently enhanced cleavage (see [E]) by Rhomboid-1-KDEL at all concentrations tested. It was thus possible to identify mutations that both enhanced and suppressed cleavage by using a Rhomboid-1-KDEL concentration that resulted in approximately 50% cleavage (25 ng).
(D) An example of the intracellular cleavage (arrow) of wild-type Spitz and each phenylalanine mutant by Rhomboid-1-KDEL.
(E) Summary of the effect of individual Spitz substrate motif mutations on cleavage by Rhomboid-1-KDEL. The top line is the wild-type Spitz substrate motif sequence (residues 138–144) plus each adjacent residue, and below it are the various point mutations that were tested. Mutations that did not affect cleavage are in black, those that mildly reduced cleavage are in gray, while those that strongly reduced cleavage are in outline. The four mutations that enhanced cleavage are in lowercase.
Molecular Cell  , DOI: ( /S (03) )

7. Figure 6 Substrate Specificity of Diverse Rhomboid Proteases
(A–C) Rhomboid proteases from (A) Drosophila, (B) vertebrate rhomboids including RHBDL-2 from humans (HsRH-2), mouse (MmRH-2) and zebrafish (DrRH-2), and (C) bacterial rhomboids from Bacillus subtilis (YqgP), Providencia stuartii (AarA), and Escherichia coli (GlpG) were tested for their ability to cleave the TMD-4/4 (−) and the TMD-4/4+GA (GA) chimeras. Presence of the GA converted this chimera into a substrate for most of these rhomboid proteases.
(D) The product resulting from P. stuartii and E. coli rhomboid cleavage was poorly secreted (Urban et al., 2002b) and was therefore examined in cells 18 hr after transfection (without media conditioning). Under these conditions, the P. stuartii rhomboid protease cleaved the chimera containing the GA motif strongly while E. coli rhomboid cleaved it less efficiently (arrow). It should be noted that the E. coli rhomboid displays only weak proteolytic activity in mammalian cells (Urban et al., 2002b). Cleavage assays were performed in the presence of Batimastat.
Molecular Cell  , DOI: ( /S (03) )

8. Figure 7
Cleavage of Toxoplasma gondii MIC Protein TMDs by Diverse Rhomboid Proteases
(A) An alignment of the Drosophila Spitz and Keren TMDs with the T. gondii MIC-2, MIC-6, and MIC-12 TMDs. The two motifs of the Spitz substrate motif and corresponding residues of the other TMDs are highlighted by outline lettering, and the predicted TMDs are boxed.
(B) Synthetic substrates containing the MIC-2, 6, and 12 TMDs were efficient substrates for Drosophila Rhomboid-1 (Rho), human RHBDL-2 (hRH), and Providencia AarA. The cleaved bands in cell lysates are indicated by arrows. Cleavage assays were performed in the presence of Batimastat, except those labeled MP, which served as positive controls for substrate expression, secretion, and cleavage by cellular metalloproteases.
Molecular Cell  , DOI: ( /S (03) )